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Dual utilization of an acceptor/donor splice site governs the alternative splicing of the IRF-3 gene
Karpova, Alla Y.; Howley, Peter M.; Ronco, Lucienne V.
Interferon regulatory factors constitute a family of transcriptional activators and repressors involved in a large number of vital cellular processes. Interferon regulatory factor-3 (IRF-3) has been implicated in virus and double-stranded RNA mediated induction of IFN? and RANTES, in DNA damage signaling, and in virus-induced apoptosis. With its critical role in these pathways, the activity of IRF-3 is tightly regulated in myriad ways. Here we describe novel regulation of IRF-3 at the level of RNA splicing. We show that an unprecedented dual utilization of a splice acceptor/donor site within the IRF-3 mRNA governs the production of two alternative splice isoforms. PMID:11090129
Analysis of donor splice sites in different eukaryotic organisms
Igor B. Rogozin; Luciano Milanesi
We present here a new algorithm for functional site analysis. It is based on four main assumptions: each variation of nucleotide\\u000a composition makes a different contribution to the overall binding free energy of interaction between a functional site and\\u000a another molecule; nonfunctioning site-like regions (pseudosites) are absent or rare in genomes; there may be errors in the\\u000a sample of sites;
Characterization of Conserved Tandem Donor Sites and Intronic Motifs Required for Alternative Splicing in Corticosteroid Receptor Genes
Qian, Xiaoxiao; Matthews, Laura; Lightman, Stafford; Ray, David; Norman, Michael
Alternative splicing events from tandem donor sites result in mRNA variants coding for additional amino acids in the DNA binding domain of both the glucocorticoid (GR) and mineralocorticoid (MR) receptors. We now show that expression of both splice variants is extensively conserved in mammalian species, providing strong evidence for their functional significance. An exception to the conservation of the MR tandem splice site (an A at position +5 of the MR+12 donor site in the mouse) was predicted to decrease U1 small nuclear RNA binding. In accord with this prediction, we were unable to detect the MR+12 variant in this species. The one exception to the conservation of the GR tandem splice site, an A at position +3 of the platypus GR? donor site that was predicted to enhance binding of U1 snRNA, was unexpectedly associated with decreased expression of the variant from the endogenous gene as well as a minigene. An intronic pyrimidine motif present in both GR and MR genes was found to be critical for usage of the downstream donor site, and overexpression of TIA1/TIAL1 RNA binding proteins, which are known to bind such motifs, led to a marked increase in the proportion of GR? and MR+12. These results provide striking evidence for conservation of a complex splicing mechanism that involves processes other than stochastic spliceosome binding and identify a mechanism that would allow regulation of variant expression. PMID:19819975
GT to AT transition at a splice donor site causes skipping of the preceding exon in phenylketonuria.
Marvit, J; DiLella, A G; Brayton, K; Ledley, F D; Robson, K J; Woo, S L
Classical Phenylketonuria (PKU) is an autosomal recessive human genetic disorder caused by a deficiency of hepatic phenylalanine hydroxylase (PAH). We isolated several mutant PAH cDNA clones from a PKU carrier individual and showed that they contained an internal 116 base pair deletion, corresponding precisely to exon 12 of the human chromosomal PAH gene. The deletion causes the synthesis of a truncated protein lacking the C-terminal 52 amino acids. Gene transfer and expression studies using the mutant PAH cDNA indicated that the deletion abolishes PAH activity in the cell as a result of protein instability. To determine the molecular basis of the deletion, the mutant chromosomal PAH gene was isolated from this individual and shown to contain a GT-- greater than AT substitution at the 5' splice donor site of intron 12. Thus, the consequence of the splice donor site mutation in the human liver is the skipping of the preceding exon during RNA splicing. Images PMID:3615198
The HIV-1 5' LTR poly(A) site is inactivated by U1 snRNP interaction with the downstream major splice donor site.
Ashe, M P; Pearson, L H; Proudfoot, N J
The inactivity of the 5' long terminal repeat (LTR) poly(A) site immediately downstream of the cap site maximizes the production of HIV-1 transcripts. In this paper, we demonstrate that this inactivity is mediated by the interaction of the U1 snRNP with the major splice donor site (MSD). The inhibition of the HIV-1 poly(A) site by U1 snRNP relies on a series of delicately balanced RNA processing signals. These include the poly(A) site, the major splice donor site and the splice acceptor sites. The inherent efficiency of the HIV-1 poly(A) site allows maximal activity where there is no donor site (in the 3' LTR) but full inhibition by the downstream MSD (in the 5' LTR). The MSD must interact efficiently with U1 snRNP to completely inhibit the 5' LTR poly(A) site, whereas the splice acceptor sites are inefficient, allowing full-length genomic RNA production. PMID:9312033
A method for identifying alternative or cryptic donor splice sites within gene and mRNA sequences. Comparisons among sequences from vertebrates, echinoderms and other groups
Buckley, Katherine M; Florea, Liliana D; Smith, L Courtney
Background As the amount of genome sequencing data grows, so does the problem of computational gene identification, and in particular, the splicing signals that flank exon borders. Traditional methods for identifying splicing signals have been created and optimized using sequences from model organisms, mostly vertebrate and yeast species. However, as genome sequencing extends across the animal kingdom and includes various invertebrate species, the need for mechanisms to recognize splice signals in these organisms increases as well. With that aim in mind, we generated a model for identifying donor and acceptor splice sites that was optimized using sequences from the purple sea urchin, Strongylocentrotus purpuratus. This model was then used to assess the possibility of alternative or cryptic splicing within the highly variable immune response gene family known as 185/333. Results A donor splice site model was generated from S. purpuratus sequences that incorporates non-adjacent dependences among positions within the 9 nt splice signal and uses position weight matrices to determine the probability that the site is used for splicing. The Purpuratus model was shown to predict splice signals better than a similar model created from vertebrate sequences. Although the Purpuratus model was able to correctly predict the true splice sites within the 185/333 genes, no evidence for alternative or trans-gene splicing was observed. Conclusion The data presented herein describe the first published analyses of echinoderm splice sites and suggest that the previous methods of identifying splice signals that are based largely on vertebrate sequences may be insufficient. Furthermore, alternative or trans-gene splicing does not appear to be acting as a diversification mechanism in the 185/333 gene family. PMID:19607703
TassDB: a database of alternative tandem splice sites
Michael Hiller; Swetlana Nikolajewa; Klaus Huse; Karol Szafranski; Philip Rosenstiel; Stefan Schuster; Rolf Backofen; Matthias Platzer
Subtle alternative splice events at tandem splice sites are frequent in eukaryotes and substantially increase the complexity of transcriptomes and proteomes. We have developed a relational data- base, TassDB (TAndem Splice Site DataBase), which stores extensive data about alternative splice events at GYNGYN donors and NAGNAG acceptors. These splice events are of subtle nature since they mostly result in the
Factor IXMadrid 2: a deletion/insertion in factor IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site.
Solera, J; Magall�n, M; Martin-Villar, J; Coloma, A
DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5' end of intron d and the two last coding nucleotides located at the 3' end of exon IV in the normal factor IX gene; this fragment has been replaced by a 47-bp sequence from the normal factor IX gene, although this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends of the deleted DNA fragment. Images Figure 1 PMID:1346483
Factor IXMadrid 2: a deletion/insertion in factor IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site.
Solera, J; Magall�n, M; Martin-Villar, J; Coloma, A
DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5' end of intron d and the two last coding nucleotides located at the 3' end of exon IV in the normal factor IX gene; this fragment has been replaced by a 47-bp sequence from the normal factor IX gene, although this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends of the deleted DNA fragment. PMID:1346483
Galactosemia caused by a point mutation that activates cryptic donor splice site in the galactose-1-phosphate uridyltransferase gene
Wadelius, C.; Lagerkvist, A. (Univ. Hospital, Uppsala (Sweden) Uppsala Univ. (Sweden)); Molin, A.K.; Larsson, A. (Univ. Hospital, Uppsala (Sweden)); Von Doebeln, U. (Karolinska Institute, Stockholm (Sweden))
Galactosemia affects 1/84,000 in Sweden and is manifested in infancy when the child is exposed to galactose in the diet. If untreated there is a risk of severe early symptoms and, even with a lactose-free diet, late symptoms such as mental retardation and ovarial dysfunction may develop. In classical galactosemia, galactose-1-phosphate uridyltransferase (GALT) (EC 188.8.131.52) is defective and the normal cDNA sequence of this enzyme has been characterized. Recently eight mutations leading to galactosemia were published. Heparinized venous blood was drawn from a patient with classical galactosemia. In the cDNA from the patient examined, an insertion of 54 bp was found at position 1087. Amplification of the relevant genomic region of the patient's DNA was performed. Exon-intron boundaries and intronic sequences thus determined revealed that the 54-bp insertion was located immediately downstream of exon 10. It was further found that the patient was heterozygous for a point mutation, changing a C to a T (in 5 of 9 clones) at the second base in the intron downstream of the insertion. This alteration creates a sequence which, as well as the ordinary splice site, differs in only two positions from the consensus sequence. It was found that the mutation occurred in only one of the 20 alleles from galactosemic patients and in none of the 200 alleles from normal controls. The mutation is inherited from the mother, who also was found to express the 54-bp-long insertion at the mRNA level. Sequences from the 5[prime] end of the coding region were determined after genomic amplification, revealing a sequence identical to that reported. The mutation on the paternal allele has not been identified. 9 refs., 1 fig.
A novel point mutation (G-1 to T) in a 5' splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker muscular dystrophy.
Hagiwara, Y.; Nishio, H.; Kitoh, Y.; Takeshima, Y.; Narita, N.; Wada, H.; Yokoyama, M.; Nakamura, H.; Matsuo, M.
The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. We now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5' splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5' splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G-1-to-T mutation at the 5' splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection. Images Figure 2 Figure 5 PMID:8279470
Factor IX[sub Madrid 2]: A deletion/insertion in Facotr IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site
Solera, J. (Unidades de Genetica Molecular, Madrid (Spain)); Magallon, M.; Martin-Villar, J. (Hemofilia Hospital, Madrid (Spain)); Coloma, A. (Departamento deBioquimica de la Facultad de Medicina de la Universidad Autonoma, Madrid (Spain))
DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5[prime] end of intron d and the two last coding nucleotides located at the 3[prime] end of exon IV in the normal factor IX gene; this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends of the deleted DNA fragment.
Structural investigation of HIV-1 genomic RNA dimerization process reveals a role for the Major Splice-site Donor stem loop.
Deforges, Jules; Chamond, Nathalie; Sargueil, Bruno
The 5'UnTranslated Region (5'UTR) of HIV-1 genomic RNA, which precedes the Gag coding sequence, fulfills several roles during the lentivirus life cycle. This 335 nucleotides leader contains many stable structures that are crucial for the regulation of genetic expression at the level of transcription, splicing, and translation. In the late steps of the virus cycle, i.e. virions formation, the genomic RNA serves as propagated genome and its encapsidation in new particles relies on its ability to form non-covalent dimers. Dimerization is proposed to be initiated by the intermolecular pairing of a self-complementary sequence located in the apical loop of the DIS hairpin (Dimer Initiation Sequence). The regulation of this phenomenon and the extraordinary stability of the dimers imply that structural elements other than this kissing complex remain to be identified. Here, we show that swapping the Gag open reading frame (ORF) by reporter genes interferes with dimers formation efficiency. Importantly, the nature of the ORF alters specific structures of the 5'UTR. By using a systematic "SHAPE" approach, we pointed out that sequences within the Major Splice Site are involved in the dimerization process. Furthermore, by the use of an antisense oligonucleotide specific for the MSD associated to a SHAPE analysis of the 5'UTR structure, we demonstrated that interfering with the MSD results both in an impaired dimerization and in modifications of the 5'UTR structure. All together these data support a recently proposed model in which intramolecular base pairings are important determinants for the dimerization process. We further conclude that much care should be taken when comparing translation activity of reporter constructs with the viral situation. PMID:22365986
A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811+1.6kbA-->G, produces a new exon: high frequency in Spanish cystic fibrosis chromosomes and association with severe phenotype.
Chill�n, M; D�rk, T; Casals, T; Gim�nez, J; Fonknechten, N; Will, K; Ramos, D; Nunes, V; Estivill, X
mRNA analysis of the cystic fibrosis transmembrane regulator (CFTR) gene in tissues of cystic fibrosis (CF) patients has allowed us to detect a cryptic exon. The new exon involves 49 base pairs between exons 11 and 12 and is due to a point mutation (1811+1.6kbA-->G) that creates a new donor splice site in intron 11. Semiquantitative mRNA analysis showed that 1811+1.6kbA-->G-mRNA was 5-10-fold less abundant than delta F508 mRNA. Mutation 1811+1.6kbA-->G was found in 21 Spanish and 1 German CF chromosomes, making it the fourth-most-frequent mutation (2%) in the Spanish population. Individuals with genotype delta F508/1811+1.6kbA-->G have only 1%-3% of normal CFTR mRNA. This loss of 97% of normal CFTR mRNA must be responsible for the pancreatic insufficiency and for the severe CF phenotype in these patients. Images Figure 3 PMID:7534040
A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811 + 1.6kbA {yields} G, produces a new exon: High frequency in spanish cystic fibrosis chromosomes and association with severe phenotype
Chillon, M.; Casals, T.; Gimenez, J.; Ramos, D.; Nunes, V.; Estivill, X. [Cancer Research Institute, Barcelona (Spain); Doerk, T.; Will, K. [Medizinische Hochschule Hannover (Germany); Fonknechten, N. [Institut Cochin de Genetique Moleculaire, Paris (France)
mRNA analysis of the cystic fibrosis transmembrane regulator (CFTR) gene in tissues of cystic fibrosis (CF) patients has allowed us to detect a cryptic exon. The new exon involves 49 base pairs between exons 11 and 12 and is due to a point mutation (1811+1.6bA{yields}G) that creates a new donor splice site in intron 11. Semiquantitative mRNA analysis showed that 1811+1.6kbA{r_arrow}G-mRNA was 5-10-fold less abundant than {triangle}F508 mRNA. Mutations 1811+1.6kbA{yields}G was found in 21 Spanish and 1 German CF chromosome(s), making it the fourth-most-frequent mutation (2%) in the Spanish population. Individuals with genotype {triangle}F508/1811+1.6kbA{yields}G have only 1%-3% of normal CFTR mRNA. This loss of 97% of normal CFTR mRNA must be responsible for the pancreatic insufficiency and for the severe CF phenotype in these patients. 30 refs., 3 figs., 2 tabs.
Alternative splicing at GYNNGY 5? splice sites: more noise, less regulation
Wang, Meng; Zhang, Peiwei; Shu, Yang; Yuan, Fei; Zhang, Yuchao; Zhou, You; Jiang, Min; Zhu, Yufei; Hu, Landian; Kong, Xiangyin; Zhang, Zhenguo
Numerous eukaryotic genes are alternatively spliced. Recently, deep transcriptome sequencing has skyrocketed proportion of alternatively spliced genes; over 95% human multi-exon genes are alternatively spliced. One fundamental question is: are all these alternative splicing (AS) events functional? To look into this issue, we studied the most common form of alternative 5? splice sites�GYNNGYs (Y = C/T), where both GYs can function as splice sites. Global analyses suggest that splicing noise (due to stochasticity of splicing process) can cause AS at GYNNGYs, evidenced by higher AS frequency in non-coding than in coding regions, in non-conserved than in conserved genes and in lowly expressed than in highly expressed genes. However, ?20% AS GYNNGYs in humans and ?3% in mice exhibit tissue-dependent regulation. Consistent with being functional, regulated GYNNGYs are more conserved than unregulated ones. And regulated GYNNGYs have distinctive sequence features which may confer regulation. Particularly, each regulated GYNNGY comprises two splice sites more resembling each other than unregulated GYNNGYs, and has more conserved downstream flanking intron. Intriguingly, most regulated GYNNGYs may tune gene expression through coupling with nonsense-mediated mRNA decay, rather than encode different proteins. In summary, AS at GYNNGY 5? splice sites is primarily splicing noise, and secondarily a way of regulation. PMID:25428370
Splicing in Caenorhabditis elegans does not require an AG at the 3' splice acceptor site.
Aroian, R V; Levy, A D; Koga, M; Ohshima, Y; Kramer, J M; Sternberg, P W
The dinucleotide AG, found at the 3' end of virtually all eukaryotic pre-mRNA introns, is thought to be essential for splicing. Reduction-of-function mutations in two Caenorhabditis elegans genes, the receptor tyrosine kinase gene let-23 and the collagen gene dpy-10, both alter the AG at the end of a short (ca. 50-nucleotide) intron to AA. The in vivo effects of these mutations were studied by sequencing polymerase chain reaction-amplified reverse-transcribed RNA isolated from the two mutants. As expected, we find transcripts that splice to a cryptic AG, skip an exon, and retain an unspliced intron. However, we also find significant levels of splicing at the mutated 3' splice site (AA) and at nearby non-AG dinucleotides. Our results indicate that for short C. elegans introns an AG is not required for splicing at either the correct 3' splice site or incorrect sites. Analysis of a splice site mutant involving a longer, 316-nucleotide C. elegans intron indicates that an AG is also not required there for splicing. We hypothesize that elements besides the invariant AG, e.g., an A-U-rich region, a UUUC motif, and/or a potential branch point sequence, are directing the selection of the 3' splice site and that in wild-type genes these elements cooperate so that proper splicing occurs. PMID:8417357
Direct selection for mutations affecting specific splice sites in a hamster dihydrofolate reductase minigene.
Chen, I T; Chasin, L A
A Chinese hamster cell line containing an extra exon 2 (50 bp) inserted into a single intron of a dihydrofolate reductase (dhfr) minigene was constructed. The extra exon 2 was efficiently spliced into the RNA, resulting in an mRNA that is incapable of coding for the DHFR enzyme. Mutations that decreased splicing of this extra exon 2 caused it to be skipped and so produced normal dhfr mRNA. In contrast to the parental cell line, the splicing mutants display a DHFR-positive growth phenotype. Splicing mutants were isolated from this cell line after treatment with four different mutagens (racemic benzo[c]phenanthrene diol epoxide, ethyl methanesulfonate, ethyl nitrosourea, and UV irradiation). By polymerase chain reaction amplification and direct DNA sequencing, we determined the base changes in 66 mutants. Each of the mutagens generated highly specific base changes. All mutations were single-base substitutions and comprised 24 different changes distributed over 16 positions. Most of the mutations were within the consensus sequences at the exon 2 splice donor, acceptor, and branch sites. The RNA splicing patterns in the mutants were analyzed by quantitative reverse transcription-polymerase chain reaction. The recruitment of cryptic sites was rarely seen; simple exon skipping was the predominant mutant phenotype. The wide variety of mutations that produced exon skipping suggests that this phenotype is the typical consequence of splice site damage and supports the exon definition model of splice site selection. A few mutations were located outside the consensus sequences, in the exon or between the branch point and the polypyrimidine tract, identifying additional positions that play a role in splice site definition. That most of these 66 mutations fell within consensus sequences in this near-saturation mutagenesis suggests that splicing signals beyond the consensus may consist of robust RNA structures. Images PMID:8417332
SpliceAid-F: a database of human splicing factors and their RNA-binding sites
Giulietti, Matteo; Piva, Francesco; D�Antonio, Mattia; D�Onorio De Meo, Paolo; Paoletti, Daniele; Castrignan�, Tiziana; D�Erchia, Anna Maria; Picardi, Ernesto; Zambelli, Federico; Principato, Giovanni; Pavesi, Giulio; Pesole, Graziano
A comprehensive knowledge of all the factors involved in splicing, both proteins and RNAs, and of their interaction network is crucial for reaching a better understanding of this process and its functions. A large part of relevant information is buried in the literature or collected in various different databases. By hand-curated screenings of literature and databases, we retrieved experimentally validated data on 71 human RNA-binding splicing regulatory proteins and organized them into a database called �SpliceAid-F� (http://www.caspur.it/SpliceAidF/). For each splicing factor (SF), the database reports its functional domains, its protein and chemical interactors and its expression data. Furthermore, we collected experimentally validated RNA�SF interactions, including relevant information on the RNA-binding sites, such as the genes where these sites lie, their genomic coordinates, the splicing effects, the experimental procedures used, as well as the corresponding bibliographic references. We also collected information from experiments showing no RNA�SF binding, at least in the assayed conditions. In total, SpliceAid-F contains 4227 interactions, 2590 RNA-binding sites and 1141 �no-binding� sites, including information on cellular contexts and conditions where binding was tested. The data collected in SpliceAid-F can provide significant information to explain an observed splicing pattern as well as the effect of mutations in functional regulatory elements. PMID:23118479
Splice site strength�dependent activity and genetic buffering by poly-G runs
Xiao, Xinshu
Pre-mRNA splicing is regulated through the combinatorial activity of RNA motifs, including splice sites and splicing regulatory elements. Here we show that the activity of the G-run (polyguanine sequence) class of splicing ...
Site-specific deletion in cauliflower mosaic virus DNA: possible involvement of RNA splicing and reverse transcription
Hirochika, Hirohiko; Takatsuji, Hiroshi; Ubasawa, Aiko; Ikeda, Joh-E
A frequent site-specific deletion was observed in the life cycle of cauliflower mosaic virus (S strain). Analysis of the sequence around the deletion site and the parental sequence implied that the deletion was promoted at sequences similar to the donor and acceptor consensus sequences of RNA splicing, designated as the deletion donor and acceptor sequences, respectively. To elucidate the mechanism of this site-specific deletion, point mutations were introduced into the deletion donor sequence (GT to GG or GA transversion). Deletion at the original deletion donor site did not occur in these mutants, instead, new (cryptic) donor sites were activated. All of these activated cryptic sites had sequences similar to the splicing consensus sequence. In all cases except one, the original deletion acceptor site was used. These results can be most readily explained by postulating that the site-specific deletion occurs by reverse transcription of spliced viral RNA. This frequent site-specific deletion was not observed in other strains. For a virus which replicates by reverse transcription, a mechanism to regulate the rate of splicing is required to ensure the intactness of the viral genome. We discuss the possibility that the S strain has a mutation in this regulatory mechanism. ImagesFig. 1.Fig. 3.Fig. 5.Fig. 7. PMID:16453624
Ex vivo splicing assays of mutations at noncanonical positions of splice sites in USHER genes.
Le Gu�dard-M�reuze, Sandie; Vach�, Christel; Baux, David; Faug�re, Val�rie; Larrieu, Lise; Abadie, Caroline; Janecke, Andreas; Claustres, Mireille; Roux, Anne-Fran�oise; Tuffery-Giraud, Sylvie
Molecular diagnosis in Usher syndrome type 1 and 2 patients led to the identification of 21 sequence variations located in noncanonical positions of splice sites in MYO7A, CDH23, USH1C, and USH2A genes. To establish experimentally the splicing pattern of these substitutions, whose impact on splicing is not always predictable by available softwares, ex vivo splicing assays were performed. The branch-point mapping strategy was also used to investigate further a putative branch-point mutation in USH2A intron 43. Aberrant splicing was demonstrated for 16 of the 21 (76.2%) tested sequence variations. The mutations resulted more frequently in activation of a nearby cryptic splice site or use of a de novo splice site than exon skipping (37.5%). This study allowed the reclassification as splicing mutations of one silent (c.7872G>A (p.Glu2624Glu) in CDH23) and four missense mutations (c.2993G>A (p.Arg998Lys) in USH2A, c.592G>A (p.Ala198Thr), c.3503G>C [p.Arg1168Pro], c.5944G>A (p.Gly1982Arg) in MYO7A), whereas it provided clues about a role in structure/function in four other cases: c.802G>A (p.Gly268Arg), c.653T>A (p.Val218Glu) (USH2A), and c.397C>T (p.His133Tyr), c.3502C>T (p.Arg1168Trp) (MYO7A). Our data provide insights into the contribution of splicing mutations in Usher genes and illustrate the need to define accurately their splicing outcome for diagnostic purposes. PMID:20052763
Exonic Splicing Enhancer-Dependent Selection of the Bovine Papillomavirus Type 1 Nucleotide 3225 3' Splice Site Can Be Rescued in a Cell Lacking Splicing Factor ASF\\/SF2 through Activation of the Phosphatidylinositol 3Kinase\\/Akt Pathway
Xuefeng Liu; Akila Mayeda; Mingfang Tao; Zhi-Ming Zheng
Bovine papillomavirus type 1 (BPV-1) late pre-mRNAs are spliced in keratinocytes in a differentiation- specific manner: the late leader 5 splice site alternatively splices to a proximal 3 splice site (at nucleotide 3225) to express L2 or to a distal 3 splice site (at nucleotide 3605) to express L1. Two exonic splicing enhancers, each containing two ASF\\/SF2 (alternative splicing factor\\/splicing
Splicing of many human genes involves sites embedded within introns.
Kelly, Steven; Georgomanolis, Theodore; Zirkel, Anne; Diermeier, Sarah; O'Reilly, Dawn; Murphy, Shona; L�ngst, Gernot; Cook, Peter R; Papantonis, Argyris
The conventional model for splicing involves excision of each intron in one piece; we demonstrate this inaccurately describes splicing in many human genes. First, after switching on transcription of SAMD4A, a gene with a 134 kb-long first intron, splicing joins the 3' end of exon 1 to successive points within intron 1 well before the acceptor site at exon 2 is made. Second, genome-wide analysis shows that >60% of active genes yield products generated by such intermediate intron splicing. These products are present at ?15% the levels of primary transcripts, are encoded by conserved sequences similar to those found at canonical acceptors, and marked by distinctive structural and epigenetic features. Finally, using targeted genome editing, we demonstrate that inhibiting the formation of these splicing intermediates affects efficient exon-exon splicing. These findings greatly expand the functional and regulatory complexity of the human transcriptome. PMID:25897131
Splicing of many human genes involves sites embedded within introns
Kelly, Steven; Georgomanolis, Theodore; Zirkel, Anne; Diermeier, Sarah; O'Reilly, Dawn; Murphy, Shona; L�ngst, Gernot; Cook, Peter R.; Papantonis, Argyris
The conventional model for splicing involves excision of each intron in one piece; we demonstrate this inaccurately describes splicing in many human genes. First, after switching on transcription of SAMD4A, a gene with a 134 kb-long first intron, splicing joins the 3? end of exon 1 to successive points within intron 1 well before the acceptor site at exon 2 is made. Second, genome-wide analysis shows that >60% of active genes yield products generated by such intermediate intron splicing. These products are present at ?15% the levels of primary transcripts, are encoded by conserved sequences similar to those found at canonical acceptors, and marked by distinctive structural and epigenetic features. Finally, using targeted genome editing, we demonstrate that inhibiting the formation of these splicing intermediates affects efficient exon�exon splicing. These findings greatly expand the functional and regulatory complexity of the human transcriptome. PMID:25897131
New Splice Site Acceptor Mutation in AIRE Gene in Autoimmune Polyendocrine Syndrome Type 1
Mora, Mireia; Hanzu, Felicia A.; Pradas-Juni, Marta; Aranda, Gloria B.; Halperin, Irene; Puig-Domingo, Manuel; Aguil�, Sira; Fern�ndez-Rebollo, Eduardo
Autoimmune polyglandular syndrome type 1 (APS-1, OMIM 240300) is a rare autosomal recessive disorder, characterized by the presence of at least two of three major diseases: hypoparathyroidism, Addison�s disease, and chronic mucocutaneous candidiasis. We aim to identify the molecular defects and investigate the clinical and mutational characteristics in an index case and other members of a consanguineous family. We identified a novel homozygous mutation in the splice site acceptor (SSA) of intron 5 (c.653-1G>A) in two siblings with different clinical outcomes of APS-1. Coding DNA sequencing revealed that this AIRE mutation potentially compromised the recognition of the constitutive SSA of intron 5, splicing upstream onto a nearby cryptic SSA in intron 5. Surprisingly, the use of an alternative SSA entails the uncovering of a cryptic donor splice site in exon 5. This new transcript generates a truncated protein (p.A214fs67X) containing the first 213 amino acids and followed by 68 aberrant amino acids. The mutation affects the proper splicing, not only at the acceptor but also at the donor splice site, highlighting the complexity of recognizing suitable splicing sites and the importance of sequencing the intron-exon junctions for a more precise molecular diagnosis and correct genetic counseling. As both siblings were carrying the same mutation but exhibited a different APS-1 onset, and one of the brothers was not clinically diagnosed, our finding highlights the possibility to suspect mutations in the AIRE gene in cases of childhood chronic candidiasis and/or hypoparathyroidism otherwise unexplained, especially when the phenotype is associated with other autoimmune diseases. PMID:24988226
Method of predicting Splice Sites based on signal interactions
Churbanov, Alexander; Rogozin, Igor B; Deogun, Jitender S; Ali, Hesham
Background Predicting and proper ranking of canonical splice sites (SSs) is a challenging problem in bioinformatics and machine learning communities. Any progress in SSs recognition will lead to better understanding of splicing mechanism. We introduce several new approaches of combining a priori knowledge for improved SS detection. First, we design our new Bayesian SS sensor based on oligonucleotide counting. To further enhance prediction quality, we applied our new de novo motif detection tool MHMMotif to intronic ends and exons. We combine elements found with sensor information using Naive Bayesian Network, as implemented in our new tool SpliceScan. Results According to our tests, the Bayesian sensor outperforms the contemporary Maximum Entropy sensor for 5' SS detection. We report a number of putative Exonic (ESE) and Intronic (ISE) Splicing Enhancers found by MHMMotif tool. T-test statistics on mouse/rat intronic alignments indicates, that detected elements are on average more conserved as compared to other oligos, which supports our assumption of their functional importance. The tool has been shown to outperform the SpliceView, GeneSplicer, NNSplice, Genio and NetUTR tools for the test set of human genes. SpliceScan outperforms all contemporary ab initio gene structural prediction tools on the set of 5' UTR gene fragments. Conclusion Designed methods have many attractive properties, compared to existing approaches. Bayesian sensor, MHMMotif program and SpliceScan tools are freely available on our web site. Reviewers This article was reviewed by Manyuan Long, Arcady Mushegian and Mikhail Gelfand. PMID:16584568
p54nrb associates with the 5? splice site within large transcription/splicing complexes
Kameoka, Sei; Duque, Paula; Konarska, Maria M
The functional coupling of transcription and splicing has been reported both in vivo and in vitro, but the molecular mechanisms governing these interactions remain largely unknown. Here we show that p54nrb, a transcription/splicing factor, associates with the 5? splice site (SS) within large complexes present in HeLa cell nuclear extracts, in which the hyperphosphorylated form of RNA polymerase II (RNAPIIO) is associated with U1 or U1 and U2 snRNPs. These RNAPIIO�snRNP complexes also contain other transcription/splicing factors, such as PSF and TLS, as well as transcription factors that interact with RNAPIIO during elongation, including P-TEFb, TAT-SF1 and TFIIF. The presence of these factors in functional elongation complexes, demonstrated using an immobilized DNA template assay, strongly suggests that the RNAPIIO�snRNP complexes reflect physiologically relevant interactions between the transcription and splicing machineries. Our finding that both p54nrb and PSF, which bind the C-terminal domain of the largest subunit of RNAPII, can interact directly with the 5? SS indicates that these factors may mediate contacts between RNAPII and snRNPs during the coupled transcription/splicing process. PMID:15057275
Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions
T. A. Thanaraj; Francis Clark
It has been previously observed that the intrinsically weak variant GC donor sites, in order to be recog- nized by the U2-type spliceosome, possess strong consensus sequences maximized for base pair formation with U1 and U5\\/U6 snRNAs. However, varia- bility in signal strength is a fundamental mechanism for splice site selection in alternative splicing. Here we report human alternative GC-AG
The Localization of Sites Containing Nascent RNA and Splicing Factors
Ana Pombo; Peter R. Cook
Sites of transcription in the HeLa nucleus are not diffusely spread throughout euchromatin but are concentrated in ?2000 discrete sites or �transcription factories.� These sites can be immunolabeled after allowing permeabilized cells to elongate nascent RNA chains in the presence of BrUTP. Splicing factors are also concentrated in a few tens of nuclear domains known as �speckles�; they are also
Whole Exome Sequencing Reveals Novel PHEX Splice Site Mutations in Patients with Hypophosphatemic Rickets
Gillies, Christopher; Sampson, Matthew G.; Kher, Vijay; Sethi, Sidharth K.; Otto, Edgar A.
Objective Hypophosphatemic rickets (HR) is a heterogeneous genetic phosphate wasting disorder. The disease is most commonly caused by mutations in the PHEX gene located on the X-chromosome or by mutations in CLCN5, DMP1, ENPP1, FGF23, and SLC34A3. The aims of this study were to perform molecular diagnostics for four patients with HR of Indian origin (two independent families) and to describe their clinical features. Methods We performed whole exome sequencing (WES) for the affected mother of two boys who also displayed the typical features of HR, including bone malformations and phosphate wasting. B-lymphoblast cell lines were established by EBV transformation and subsequent RT-PCR to investigate an uncommon splice site variant found by WES. An in silico analysis was done to obtain accurate nucleotide frequency occurrences of consensus splice positions other than the canonical sites of all human exons. Additionally, we applied direct Sanger sequencing for all exons and exon/intron boundaries of the PHEX gene for an affected girl from an independent second Indian family. Results WES revealed a novel PHEX splice acceptor mutation in intron 9 (c.1080-3C>A) in a family with 3 affected individuals with HR. The effect on splicing of this mutation was further investigated by RT-PCR using RNA obtained from a patient�s EBV-transformed lymphoblast cell line. RT-PCR revealed an aberrant splice transcript skipping exons 10-14 which was not observed in control samples, confirming the diagnosis of X-linked dominant hypophosphatemia (XLH). The in silico analysis of all human splice sites adjacent to all 327,293 exons across 81,814 transcripts among 20,345 human genes revealed that cytosine is, with 64.3%, the most frequent nucleobase at the minus 3 splice acceptor position, followed by thymidine with 28.7%, adenine with 6.3%, and guanine with 0.8%. We generated frequency tables and pictograms for the extended donor and acceptor splice consensus regions by analyzing all human exons. Direct Sanger sequencing of all PHEX exons in a sporadic case with HR from the Indian subcontinent revealed an additional novel PHEX mutation (c.1211_1215delACAAAinsTTTACAT, p.Asp404Valfs*5, de novo) located in exon 11. Conclusions Mutation analyses revealed two novel mutations and helped to confirm the clinical diagnoses of XLH in two families from India. WES helped to analyze all genes implicated in the underlying disease complex. Mutations at splice positions other than the canonical key sites need further functional investigation to support the assertion of pathogenicity. PMID:26107949
High-affinity hnRNP A1 binding sites and duplex-forming inverted repeats have similar effects on 5' splice site selection in support of a common looping out and repression mechanism.
Nasim, Faiz-Ul Hassan; Hutchison, Stephen; Cordeau, M�lanie; Chabot, Benoit
High-affinity binding sites for the hnRNP A1 protein stimulate the use of a distal 5' splice site in mammalian pre-mRNAs. Notably, strong A1-mediated shifts in splice site selection are not accompanied by equivalent changes in the assembly of U1 snRNP-containing complexes on competing 5' splice sites. To explain the above results, we have proposed that an interaction between hnRNP A1 molecules bound to high-affinity sites loops out the internal 5' splice site. Here, we present additional evidence in support of the looping out model. First, replacing A1 binding sites with sequences that can generate a loop through RNA duplex formation activates distal 5' splice site usage in an equivalent manner. Second, increasing the distance between the internal 5' splice site and flanking A1 binding sites does not compromise activation of the distal 5' splice site. Similar results were obtained with pre-mRNAs carrying inverted repeats. Using a pre-mRNA containing only one 5' splice site, we show that splicing is repressed when flanked by two high-affinity A1 binding sites or by inverted repeats, and that inactivation of the internal 5' splice site is sufficient to elicit a strong increase in the use of the distal donor site. Our results are consistent with the view that the binding of A1 to high-affinity sites promotes loop formation, an event that would repress the internal 5' splice site and lead to distal 5' splice site activation. PMID:12212851
Structural features of a 3' splice site in influenza a.
Chen, Jonathan L; Kennedy, Scott D; Turner, Douglas H
Influenza A is an RNA virus with a genome of eight negative sense segments. Segment 7 mRNA contains a 3' splice site for alternative splicing to encode the essential M2 protein. On the basis of sequence alignment and chemical mapping experiments, the secondary structure surrounding the 3' splice site has an internal loop, adenine bulge, and hairpin loop when it is in the hairpin conformation that exposes the 3' splice site. We report structural features of a three-dimensional model of the hairpin derived from nuclear magnetic resonance spectra and simulated annealing with restrained molecular dynamics. Additional insight was provided by modeling based on (1)H chemical shifts. The internal loop containing the 3' splice site has a dynamic guanosine and a stable imino (cis Watson-Crick/Watson-Crick) GA pair. The adenine bulge also appears to be dynamic with the A either stacked in the stem or forming a base triple with a Watson-Crick GC pair. The hairpin loop is a GAAA tetraloop closed by an AC pair. PMID:25909229
Structural Features of a 3? Splice Site in Influenza A
Influenza A is an RNA virus with a genome of eight negative sense segments. Segment 7 mRNA contains a 3? splice site for alternative splicing to encode the essential M2 protein. On the basis of sequence alignment and chemical mapping experiments, the secondary structure surrounding the 3? splice site has an internal loop, adenine bulge, and hairpin loop when it is in the hairpin conformation that exposes the 3? splice site. We report structural features of a three-dimensional model of the hairpin derived from nuclear magnetic resonance spectra and simulated annealing with restrained molecular dynamics. Additional insight was provided by modeling based on 1H chemical shifts. The internal loop containing the 3? splice site has a dynamic guanosine and a stable imino (cis Watson�Crick/Watson�Crick) GA pair. The adenine bulge also appears to be dynamic with the A either stacked in the stem or forming a base triple with a Watson�Crick GC pair. The hairpin loop is a GAAA tetraloop closed by an AC pair. PMID:25909229
A comprehensive survey of non-canonical splice sites in the human transcriptome
Parada, Guillermo E.; Munita, Roberto; Cerda, Cledi A.; Gysling, Katia
We uncovered the diversity of non-canonical splice sites at the human transcriptome using deep transcriptome profiling. We mapped a total of 3.7 billion human RNA-seq reads and developed a set of stringent filters to avoid false non-canonical splice site detections. We identified 184 splice sites with non-canonical dinucleotides and U2/U12-like consensus sequences. We selected 10 of the herein identified U2/U12-like non-canonical splice site events and successfully validated 9 of them via reverse transcriptase-polymerase chain reaction and Sanger sequencing. Analyses of the 184 U2/U12-like non-canonical splice sites indicate that 51% of them are not annotated in GENCODE. In addition, 28% of them are conserved in mouse and 76% are involved in alternative splicing events, some of them with tissue-specific alternative splicing patterns. Interestingly, our analysis identified some U2/U12-like non-canonical splice sites that are converted into canonical splice sites by RNA A-to-I editing. Moreover, the U2/U12-like non-canonical splice sites have a differential distribution of splicing regulatory sequences, which may contribute to their recognition and regulation. Our analysis provides a high-confidence group of U2/U12-like non-canonical splice sites, which exhibit distinctive features among the total human splice sites. PMID:25123659
Comparative analysis of sequence features involved in the recognition of tandem splice sites
Ralf Bortfeldt; Stefanie Schindler; Karol Szafranski; Stefan Schuster; Dirk Holste
BACKGROUND: The splicing of pre-mRNAs is conspicuously often variable and produces multiple alternatively spliced (AS) isoforms that encode different messages from one gene locus. Computational studies uncovered a class of highly similar isoforms, which were related to tandem 5'-splice sites (5'ss) and 3'-splice sites (3'ss), yet with very sparse anecdotal evidence in experimental studies. To compare the types and levels
A phosphorothioate at the 3' splice-site inhibits the second splicing step in a group I intron.
Suh, E; Waring, R B
RNA polymerases can synthesize RNA containing phosphorothioate linkages in which a sulfur replaces one of the nonbridging oxygens. Only the Rp isomer is generated during transcription. A Rp phosphorothioate at the 5' splice-site of the Tetrahymena group I intron does not inhibit splicing (McSwiggen, J.A. and Cech, T.R. (1989) Science 244, 679). Transcription of mutants in which the first base of the 3' exon, U+1, was mutated to C or G, in the presence, respectively, of either cytosine or guanosine thiotriphosphate, introduced a phosphorothioate at the 3' splice-site. In both cases exon ligation was blocked. In the phosphorothioate substituted U+1G mutant, a new 3' splice-site was selected one base downstream of the correct site; despite the fact that the correct site was selected with very high fidelity in unsubstituted RNA. In contrast, the exon ligation reaction was successfully performed in reverse using unsubstituted intron RNA and ligated exons containing an Rp phosphorothioate at the exon junction site. Chirality was reversed during transesterification as in 5' splice-site cleavage (vide supra). This suggests that one non-bridging oxygen is particularly crucial for both splicing reactions. Images PMID:1282238
An Exonic Splicing Silencer Downstream of the 3' Splice Site A2 Is Required for Efficient Human Immunodeficiency Virus Type 1 Replication
Joshua M. Madsen; C. Martin Stoltzfus
Alternative splicing of the human immunodeficiency virus type 1 (HIV-1) genomic mRNA produces more than 40 unique viral mRNA species, of which more than half remain incompletely spliced within an HIV-1- infected cell. Regulation of splicing at HIV-1 3 splice sites (3ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation of splicing occurs through binding of cellular factors to
Genetic diagnosis of familial hypercholesterolaemia: the importance of functional analysis of potential splice-site mutations.
Bourbon, M; Duarte, M A; Alves, A C; Medeiros, A M; Marques, L; Soutar, A K
Familial hypercholesterolemia (FH) results from defective low-density lipoprotein receptor (LDLR) activity, mainly due to LDLR gene defects. Of the many different LDLR mutations found in patients with FH, about 6% of single base substitutions are located near or within introns, and are predicted to result in exon skipping, retention of an intron, or activation of cryptic sites during mRNA splicing. This paper reports on the Portuguese FH Study, which found 10 such mutations, 6 of them novel. For the mutations that have not been described before or those whose effect on function have not been analysed, their effect on splicing was investigated, using reverse transcriptase PCR analysis of LDLR mRNA from freshly isolated blood mononuclear cells. Two of these variants (c.313+6 T-->C, c.2389G-->T (p.V776L)) caused exon skipping, and one caused retention of an intron (c.1359-5C-->G), whereas two others (c.2140+5 G-->A and c.1061-8T-->C) had no apparent effect. Any effect of c.1185G-->C (p.V374V) on splicing could not be determined because it was on an allele with a promoter mutation (-42C-->G) that was probably not transcribed. Variants in four patients lost to follow-up could not be tested experimentally, but they almost certainly affect splicing because they disrupt the invariant AG or GT in acceptor (c.818-2A-->G) or donor (c.1060+1G-->A, c.1845+1delG and c.2547+1G-->A) spice sites. These findings emphasise that care must be taken before reporting the presence or absence of a splice-site mutation in the LDLR gene for diagnostic purposes. The study also shows that relatively simple, quick and inexpensive RNA assays can evaluate putative splicing mutations that are not always predictable by available software, thereby reducing genetic misdiagnosis of patients with FH. PMID:19411563
Impairment of alternative splice sites defining a novel gammaretroviral exon within gag modifies the oncogenic properties of Akv murine leukemia virus
S�rensen, Annette Balle; Lund, Anders H; Kunder, Sandra; Quintanilla-Martinez, Leticia; Schmidt, J�rg; Wang, Bruce; Wabl, Matthias; Pedersen, Finn Skou
Background Mutations of an alternative splice donor site located within the gag region has previously been shown to broaden the pathogenic potential of the T-lymphomagenic gammaretrovirus Moloney murine leukemia virus, while the equivalent mutations in the erythroleukemia inducing Friend murine leukemia virus seem to have no influence on the disease-inducing potential of this virus. In the present study we investigate the splice pattern as well as the possible effects of mutating the alternative splice sites on the oncogenic properties of the B-lymphomagenic Akv murine leukemia virus. Results By exon-trapping procedures we have identified a novel gammaretroviral exon, resulting from usage of alternative splice acceptor (SA') and splice donor (SD') sites located in the capsid region of gag of the B-cell lymphomagenic Akv murine leukemia virus. To analyze possible effects in vivo of this novel exon, three different alternative splice site mutant viruses, mutated in either the SA', in the SD', or in both sites, respectively, were constructed and injected into newborn inbred NMRI mice. Most of the infected mice (about 90%) developed hematopoietic neoplasms within 250 days, and histological examination of the tumors showed that the introduced synonymous gag mutations have a significant influence on the phenotype of the induced tumors, changing the distribution of the different types as well as generating tumors of additional specificities such as de novo diffuse large B cell lymphoma (DLBCL) and histiocytic sarcoma. Interestingly, a broader spectrum of diagnoses was made from the two single splice-site mutants than from as well the wild-type as the double splice-site mutant. Both single- and double-spliced transcripts are produced in vivo using the SA' and/or the SD' sites, but the mechanisms underlying the observed effects on oncogenesis remain to be clarified. Likewise, analyses of provirus integration sites in tumor tissues, which identified 111 novel RISs (retroviral integration sites) and 35 novel CISs (common integration sites), did not clearly point to specific target genes or pathways to be associated with specific tumor diagnoses or individual viral mutants. Conclusion We present here the first example of a doubly spliced transcript within the group of gammaretroviruses, and we show that mutation of the alternative splice sites that define this novel RNA product change the oncogenic potential of Akv murine leukemia virus. PMID:17617899
Enhancer elements activate the weak 3' splice site of alpha-tropomyosin exon 2.
Dye, B T; Buvoli, M; Mayer, S A; Lin, C H; Patton, J G
We have identified four purine-rich sequences that act as splicing enhancer elements to activate the weak 3' splice site of alpha-tropomyosin exon 2. These elements also activate the splicing of heterologous substrates containing weak 3' splice sites or mutated 5' splice sites. However, they are unique in that they can activate splicing whether they are placed in an upstream or downstream exon, and the two central elements can function regardless of their position relative to one another. The presence of excess RNAs containing these enhancers could effectively inhibit in vitro pre-mRNA splicing reactions in a substrate-dependent manner and, at lower concentrations of competitor RNA, the addition of SR proteins could relieve the inhibition. However, when extracts were depleted by incubation with biotinylated exon 2 RNAs followed by passage over streptavidin agarose, SR proteins were not sufficient to restore splicing. Instead, both SR proteins and fractions containing a 110-kD protein were necessary to rescue splicing. Using gel mobility shift assays, we show that formation of stable enhancer-specific complexes on alpha-tropomyosin exon 2 requires the presence of both SR proteins and the 110-kD protein. By analogy to the doublesex exon enhancer elements in Drosophila, our results suggest that assembly of mammalian exon enhancer complexes requires both SR and non-SR proteins to activate selection of weak splice sites. PMID:9848651
Mutations in U6 snRNA that Alter Splice Site Specificity: Implications for the Active Site
Cammie F. Lesser; Christine Guthrie
What determines the precise sites of cleavage in the two transesterification reactions of messenger RNA (mRNA) splicing is a major unsolved question. Mutation of the invariant G (guanosine) at position 5 of 5' splice sites in Saccharomyces cerevisiae introns activates cleavage at nearby aberrant sites. A genetic approach was used to test the hypothesis that a base-pairing interaction between the
Modulation of msl-2 5' splice site recognition by Sex-lethal.
F�rch, P; Merendino, L; Mart�nez, C; Valc�rcel, J
The protein Sex-lethal (SXL) controls dosage compensation in Drosophila by inhibiting splicing and subsequently translation of male-specific-lethal-2 (msl-2) transcripts. We have previously shown that SXL blocks the binding of U2 auxiliary factor (U2AF) to the polypyrimidine (Py)-tract associated with the 3' splice site of the regulated intron. We now report that a second pyrimidine-rich sequence containing 11 consecutive uridines immediately downstream from the 5' splice site is required for efficient splicing inhibition by SXL. Psoralen-mediated crosslinking experiments suggest that SXL binding to this uridine-rich sequence inhibits recognition of the 5' splice site by U1 snRNP in HeLa nuclear extracts. We also show that SXL interferes with the binding of the protein TIA-1 to the uridine-rich stretch. Because TIA-1 binding to this sequence is necessary for U1 snRNP recruitment to msl-25' splice site and for splicing of this pre-mRNA, we propose that SXL antagonizes TIA-1 activity and thus prevents 5' splice site recognition by U1 snRNP. Taken together with previous data, we conclude that efficient retention of msl-2 intron involves inhibition of early recognition of both splice sites by SXL. PMID:11565743
The localization of sites containing nascent RNA and splicing factors.
Pombo, A; Cook, P R
Sites of transcription in the HeLa nucleus are not diffusely spread throughout euchromatin but are concentrated in approximately 2000 discrete sites or "transcription factories." These sites can be immunolabeled after allowing permeabilized cells to elongate nascent RNA chains in the presence of BrUTP. Splicing factors are also concentrated in a few tens of nuclear domains known as "speckles"; they are also more diffusely spread throughout euchromatin. As there has been some controversy whether the speckles are sites of transcription, we investigated the relative distributions of transcription factories and the speckles (detected using Sm autoimmune antibodies). We used conditions that minimize the redistribution of both the nascent RNA and the antigens during permeabilization and fixation and imaged the sites with high resolution using cryosections and a "confocal" microscope. The speckles contained little-if any-nascent RNA and so were usually not transcriptionally active, but they were often surrounded by a few transcription sites. Whether they are active sites of processing of RNA or merely sites where the machinery is stored remains to be established. PMID:8986598
Mechanisms for U2AF to define 3? splice sites and regulate alternative splicing in the human genome
Huang, Jie; Tang, Peng; Zhou, Yu; Zhou, Jie; Qiu, Jinsong; Jiang, Li; Li, Hairi; Chen, Geng; Sun, Hui; Zhang, Yi; Denise, Alain; Zhang, Dong-Er; Fu, Xiang-Dong
The U2AF heterodimer has been well studied for its role in defining functional 3? splice sites in pre-mRNA splicing, but many fundamental questions still remain unaddressed regarding the function of U2AF in mammalian genomes. Through genome-wide analysis of U2AF-RNA interactions, we report that U2AF has the capacity to directly define ~88% of functional 3? splice sites in the human genome, but numerous U2AF binding events also occur in intronic locations. Mechanistic dissection reveals that upstream intronic binding events interfere with the immediate downstream 3? splice site associated either with the alternative exon, to cause exon skipping, or with the competing constitutive exon, to induce exon inclusion. We further demonstrate partial functional impairment with leukemia-associated mutations in U2AF35, but not U2AF65, in regulated splicing. These findings reveal the genomic function and regulatory mechanism of U2AF in both normal and disease states. PMID:25326705
The Emergence of Alternative 39 and 59 Splice Site Exons from Constitutive Exons
Ast, Gil
The Emergence of Alternative 39 and 59 Splice Site Exons from Constitutive Exons Eli Koren, Galit Lev-Maor, Gil Ast* Department of Human Molecular Genetics, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Alternative 39 and 59 splice site (ss) events constitute a significant part
Logitlinear models for the prediction of splice sites in plant pre-mRNA sequences
J�rgen Kleffe; Klaus Hermann; Wolfgang Vahrson; Burkhardt Wittig; Volker Brendel
Pre-mRNA splicing in plants, while generally similar to the processes in vertebrates and yeast, is thought to involve plant specific cis-acting elements. Both mono- cot and dicot introns are typically strongly enriched in U nucleotides, and AU- or U-rich segments are thought to be involved in intron recognition, splice site selec- tion, and splicing efficiency. We have applied logit- linear
On splice site prediction using weight array models: a comparison of smoothing techniques
NASA Astrophysics Data System (ADS)
Taher, Leila; Meinicke, Peter; Morgenstern, Burkhard
In most eukaryotic genes, protein-coding exons are separated by non-coding introns which are removed from the primary transcript by a process called "splicing". The positions where introns are cut and exons are spliced together are called "splice sites". Thus, computational prediction of splice sites is crucial for gene finding in eukaryotes. Weight array models are a powerful probabilistic approach to splice site detection. Parameters for these models are usually derived from m-tuple frequencies in trusted training data and subsequently smoothed to avoid zero probabilities. In this study we compare three different ways of parameter estimation for m-tuple frequencies, namely (a) non-smoothed probability estimation, (b) standard pseudo counts and (c) a Gaussian smoothing procedure that we recently developed.
Alternative Splicing at a NAGNAG Acceptor Site as a Novel Phenotype Modifier
Alexandre Hinzpeter; Abdel Aissat; Elvira Sondo; Catherine Costa; Nicole Arous; Christine Gameiro; Natacha Martin; Agathe Tarze; Laurence Weiss; Alix de Becdeli�vre; Bruno Costes; Michel Goossens; Luis J. Galietta; Emmanuelle Girodon; Pascale Fanen
Approximately 30% of alleles causing genetic disorders generate premature termination codons (PTCs), which are usually associated with severe phenotypes. However, bypassing the deleterious stop codon can lead to a mild disease outcome. Splicing at NAGNAG tandem splice sites has been reported to result in insertion or deletion (indel) of three nucleotides. We identified such a mechanism as the origin of
Physiological state co-regulates thousands of mammalian mRNA splicing events at tandem splice sites and alternative exons
Szafranski, Karol; Fritsch, Claudia; Schumann, Frank; Siebel, Lisa; Sinha, Rileen; Hampe, Jochen; Hiller, Michael; Englert, Christoph; Huse, Klaus; Platzer, Matthias
Thousands of tandem alternative splice sites (TASS) give rise to mRNA insertion/deletion variants with small size differences. Recent work has concentrated on the question of biological relevance in general, and the physiological regulation of TASS in particular. We have quantitatively studied 11 representative TASS cases in comparison to one mutually exclusive exon case and two cassette exons (CEs) using a panel of human and mouse tissues, as well as cultured cell lines. Tissues show small but significant differences in TASS isoform ratios, with a variance 4- to 20-fold lower than seen for CEs. Remarkably, in cultured cells, all studied alternative splicing (AS) cases showed a cell-density-dependent shift of isoform ratios with similar time series profiles. A respective genome-wide co-regulation of TASS splicing was shown by next-generation mRNA sequencing data. Moreover, data from human and mouse organs indicate that this co-regulation of TASS occurs in vivo, with brain showing the strongest difference to other organs. Together, the results indicate a physiological AS regulation mechanism that functions almost independently from the splice site context and sequence. PMID:25030907
AG-dependent 3?-splice sites are predisposed to aberrant splicing due to a mutation at the first nucleotide of an exon
Fu, Yuan; Masuda, Akio; Ito, Mikako; Shinmi, Jun; Ohno, Kinji
In pre-mRNA splicing, a conserved AG/G at the 3?-splice site is recognized by U2AF35. A disease-causing mutation abrogating the G nucleotide at the first position of an exon (E+1) causes exon skipping in GH1, FECH and EYA1, but not in LPL or HEXA. Knockdown of U2AF35 enhanced exon skipping in GH1 and FECH. RNA-EMSA revealed that wild-type FECH requires U2AF35 but wild-type LPL does not. A series of artificial mutations in the polypyrimidine tracts of GH1, FECH, EYA1, LPL and HEXA disclosed that a stretch of at least 10�15 pyrimidines is required to ensure normal splicing in the presence of a mutation at E+1. Analysis of nine other disease-causing mutations at E+1 detected five splicing mutations. Our studies suggest that a mutation at the AG-dependent 3?-splice site that requires U2AF35 for spliceosome assembly causes exon skipping, whereas one at the AG-independent 3?-splice site that does not require U2AF35 gives rise to normal splicing. The AG-dependence of the 3?-splice site that we analyzed in disease-causing mutations at E+1 potentially helps identify yet unrecognized splicing mutations at E+1. PMID:21288883
Novel mitofusin 2 splice-site mutation causes Charcot-Marie-Tooth disease type 2 with prominent sensory dysfunction.
Martikainen, Mika H; Kyt�vuori, Laura; Majamaa, Kari
MFN2 mutations are a major cause of the axonal form of Charcot-Marie-Tooth disease (CMT2). MFN2 encodes mitofusin 2, a mitochondrial fusion protein that is critical for mitochondrial DNA integrity and function. Here we describe CMT2 in a Finnish man and his son, with disease onset in young adulthood, slow progression, and prominent sensory as well as autonomic dysfunction. Molecular analysis revealed in both subjects a previously unreported heterozygous MFN2 mutation c.708G>A that is predicted to abolish a donor splice site for exon 7 of the MFN2 gene. An incorrectly spliced transcript without exon 7 was detected in RT-PCR analysis. The lack of exon 7 creates frameshift and, consequently, premature termination within exon 8. We demonstrated the presence of the aberrant mRNA suggesting either dominant-negative or toxic gain-of-function effect of the heterozygous c.708G>A mutation. This novel mutation adds to the few previously reported pathogenic MFN2 splice site mutations causing CMT2. PMID:24530046
3' Splice site sequences of spinal muscular atrophy related SMN2 pre-mRNA include enhancers for nearby exons.
Cho, Sunghee; Moon, Heegyum; Loh, Tiing Jen; Oh, Hyun Kyung; Kim, Hey-Ran; Shin, Myung-Geun; Liao, D Joshua; Zhou, Jianhua; Zheng, Xuexiu; Shen, Haihong
Spinal muscular atrophy (SMA) is a human genetic disease which occurs because of the deletion or mutation of SMN1 gene. SMN1 gene encodes the SMN protein which plays a key role in spliceosome assembly. Although human patients contain SMN2, a duplicate of SMN1, splicing of SMN2 produces predominantly exon 7 skipped isoform. In order to understand the functions of splice site sequences on exon 7 and 8, we analyzed the effects of conserved splice site sequences on exon 7 skipping of SMN2 and SMN1 pre-mRNA. We show here that conserved 5' splice site sequence of exon 7 promoted splicing of nearby exons and subsequently reduced splicing of distant exons. However, to our surprise, conserved 3' splice site sequence of exon 7 and 8 did not promote splicing of nearby exons. By contrast, the mutation inhibited splicing of nearby exons and subsequently promoted splicing of distant exons. Our study shows that 3' splice sites of exon 7 and 8 contain enhancer for their splice site selection, in addition to providing cleavage sites. PMID:24616638
Clinical presentation and molecular identification of four uncommon alpha globin variants in Thailand. Initiation codon mutation of ?2-globin Gene (HBA2:c.1delA), donor splice site mutation of ?1-globin gene (IVSI-1, HBA1:c.95 + 1G>A), hemoglobin Queens Park/Chao Pra Ya (HBA1:c.98T>A) and hemoglobin Westmead (HBA2:c.369C>G).
Viprakasit, Vip; Ekwattanakit, Supachai; Chalaow, Nipon; Riolueang, Suchada; Wijit, Sirirat; Tanyut, Porntep; Chat-Uthai, Nunthawut; Tachavanich, Kalaya
Alpha thalassemia is the most common genetic disease in the world with the prevalence of carriers ranging from 5-50% in several populations. Coinheritance of two defective ?-globin genes usually gives rise to a symptomatic condition, hemoglobin (Hb) H disease. Previously, it has been suggested from several studies in different populations that nondeletional Hb H disease (--/?(T)? or --/??(T)) is generally more severe than the deletional type (--/-?). In this report, we describe four rare nondeletional ?-thalassemia mutations in Thai individuals, including initiation codon mutation (HBA2:c.1delA), donor splice site mutation (IVSI-1, HBA1:c.95 + 1G>A), Hb Queens Park (HBA1:c.98T>A) [?32(B13)Met>Lys], and Hb Westmead (HBA2:c.369C>G) [?122(H5)His>Gln]. Interactions of the first three mutations with the ?(0)-thalassemia resulted in nondeletional Hb H disease; however, their clinical presentations were rather mild and some were detected accidentally. This suggests that a genotype-phenotype correlation of ?-thalassemia syndrome might be more heterogeneous and so the type of mutation does not simply imply the prediction of the resulting phenotype. Our data will be of use in future genetic counseling of such conditions that are increasingly identified thanks to the improvement of molecular analysis in routine laboratories. PMID:24081251
Cauliflower mosaic virus Transcriptome Reveals a Complex Alternative Splicing Pattern
Bouton, Cl�ment; Geldreich, Ang�le; Ramel, La�titia; Ryabova, Lyubov A.; Dimitrova, Maria; Keller, Mario
The plant pararetrovirus Cauliflower mosaic virus (CaMV) uses alternative splic-ing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5� region and sug-gested that the main role of CaMV splicing is to downregulate expression of open read-ing frames (ORFs) I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern, as we identify new splice donor and acceptor sites whose selection leads to more than thirteen 35S RNA isoforms in infected turnip plants. Inactivating splice donor or acceptor sites is not lethal for the virus, since disrupted sites are systematically rescued by the activation of cryptic and/or seldom used splice sites. Taken together, our data depict a conserved, complex and flexible process, involving multiple sites, that ensures splicing of 35S RNA. PMID:26162084
Activation of cryptic 3' splice sites within introns of cellular genes following gene entrapment
Anna B. Osipovich; Erica K. White-Grindley; Geoffrey G. Hicks; Michael J. Roshon; Christian Shaffer; Jason H. Moore; H. Earl Ruley
Gene trap vectors developed for genome-wide muta- genesis can be used to study factors governing the expression of exons inserted throughout the genome. For example, entrapment vectors consist- ing of a partial 3�-terminal exon (i.e. a neomycin resistance gene (Neo), a poly(A) site, but no 3� splice site) were typically expressed following insertion into introns, from cellular transcripts that spliced
RNA Splicing at Human Immunodeficiency Virus Type 1 3' Splice Site A2 Is Regulated by Binding of hnRNP A\\/B Proteins to an Exonic Splicing Silencer Element
The synthesis of human immunodeficiency virus type 1 (HIV-1) mRNAs is a complex process by which more than 30 different mRNA species are produced by alternative splicing of a single primary RNA transcript. HIV-1 splice sites are used with significantly different efficiencies, resulting in different levels of mRNA species in infected cells. Splicing of Tat mRNA, which is present at
Solution structure of the pseudo-5? splice site of a retroviral splicing suppressor
Control of Rous sarcoma virus RNA splicing depends in part on the interaction of U1 and U11 snRNPs with an intronic RNA element called the negative regulator of splicing (NRS). A 23mer RNA hairpin (NRS23) of the NRS directly binds U1 and U11 snRNPs. Mutations that disrupt base-pairing between the loop of NRS23 and U1 snRNA abolish its negative control of splicing. We have determined the solution structure of NRS23 using NOEs, torsion angles, and residual dipolar couplings that were extracted from multidimensional heteronuclear NMR spectra. Our structure showed that the 6-bp stem of NRS23 adopts a nearly A-form duplex conformation. The loop, which consists of 11 residues according to secondary structure probing, was in a closed conformation. U913, the first residue in the loop, was bulged out or dynamic, and loop residues G914�C923, G915�U922, and U916�A921 were base-paired. The remaining UUGU tetraloop sequence did not adopt a stable structure and appears flexible in solution. This tetraloop differs from the well-known classes of tetraloops (GNRA, CUYG, UNCG) in terms of its stability, structure, and function. Deletion of the bulged U913, which is not complementary to U1 snRNA, increased the melting temperature of the RNA hairpin. This hyperstable hairpin exhibited a significant decrease in binding to U1 snRNP. Thus, the structure of the NRS RNA, as well as its sequence, is important for interaction with U1 snRNP and for splicing suppression. PMID:15317975
Hepatitis B Virus DNA Splicing in Lebanese Blood Donors and Genotype A to E Strains: Implications for Hepatitis B Virus DNA Quantification and Infectivity
El Chaar, Mira; El Jisr, Tamima
Hepatitis B virus (HBV) is one of the major viruses transmissible by blood that causes chronic infection in immunocompromised individuals. The study of 61 HBV carrier blood donors from Lebanon revealed multiple patterns of spliced HBV DNA. HBV DNA splicing was examined and quantified in samples of five genotypes and in seroconversion panels. The Lebanese sample median viral load was 1.5 �102 IU/ml. All strains were genotype D, serotype ayw; 35 clustered as subgenotype D1 and 7 clustered as subgenotype D2. Three splice variants (SP1, SP1A, and Pol/S) were observed in 12 high-viral-load samples. Twenty samples of each genotype, A to E, were tested for the presence of HBV spliced DNA and SP1-specific splice variant. An unspliced HBV genome was dominant, but 100% of strains with a viral load of ?105 copies/ml contained various proportions of spliced DNA. SP1 was detected in 56/100 (56%) samples in levels that correlated with the overall viral load. HBV DNA quantification with S (unspliced) and X (total DNA) regions provided different levels of viral load, with the difference corresponding to spliced DNA. During the highly infectious window period, the SP1 variant became detectable shortly after the hepatitis B surface antigen (HBsAg), suggesting a correlation between the initiation of splicing and the production of detectable levels of HBsAg. The quantification of HBV DNA with primers located outside and inside the spliced region might provide different estimations of viral load and differentiate between infectious and defective viral genomes. The role of splicing neoproteins in HBV replication and interaction with the host remains to be determined. PMID:22785194
The strength of the HIV1 3' splice sites affects Rev function
Susanne Kammler; Marianne Otte; Ilona Hauber; J�rgen Kjems; Joachim Hauber; Heiner Schaal
BACKGROUND: The HIV-1 Rev protein is a key component in the early to late switch in HIV-1 splicing from early intronless (e.g. tat, rev) to late intron-containing Rev-dependent (e.g. gag, vif, env) transcripts. Previous results suggested that cis-acting sequences and inefficient 5' and 3' splice sites are a prerequisite for Rev function. However, we and other groups have shown that
Exon 9 of the CFTR gene: splice site haplotypes and cystic fibrosis mutations
Thilo D�rk; Rainer Fislage; Thomas Neumann; Brigitte Wulf; Burkhard T�mmler
The alternatively spliced exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene codes for the initial part of the amino-terminal nucleotide-binding fold of CFTR. A unique feature of the acceptor splice site preceding this exon is a variable length polymorphism within the polypyrimidine tract influencing the extent of exon 9 skipping in CFTR mRNA. We investigated this repeat
Cross-kingdom patterns of alternative splicing and splice recognition
McGuire, Abigail M; Pearson, Matthew D; Neafsey, Daniel E; Galagan, James E
Background Variations in transcript splicing can reveal how eukaryotes recognize intronic splice sites. Retained introns (RIs) commonly appear when the intron definition (ID) mechanism of splice site recognition inconsistently identifies intron-exon boundaries, and cassette exons (CEs) are often caused by variable recognition of splice junctions by the exon definition (ED) mechanism. We have performed a comprehensive survey of alternative splicing across 42 eukaryotes to gain insight into how spliceosomal introns are recognized. Results All eukaryotes we studied exhibit RIs, which appear more frequently than previously thought. CEs are also present in all kingdoms and most of the organisms in our analysis. We observe that the ratio of CEs to RIs varies substantially among kingdoms, while the ratio of competing 3' acceptor and competing 5' donor sites remains nearly constant. In addition, we find the ratio of CEs to RIs in each organism correlates with the length of its introns. In all 14 fungi we examined, as well as in most of the 9 protists, RIs far outnumber CEs. This differs from the trend seen in 13 multicellular animals, where CEs occur much more frequently than RIs. The six plants we analyzed exhibit intermediate proportions of CEs and RIs. Conclusion Our results suggest that most extant eukaryotes are capable of recognizing splice sites via both ID and ED, although ED is most common in multicellular animals and ID predominates in fungi and most protists. PMID:18321378
Familial glucocorticoid resistance caused by a splice site deletion in the human glucocorticoid receptor gene
Karl, M.; Lamberts, S.W.J.; Detera-Wadleigh, S.D.; Encio, I.J.; Stratakis, C.A.; Hurley, D.M.; Accili, D.; Chrousos, G.P. (National Institutes of Health, Bethesda, MD (United States) Erasmus Univ. of Rotterdam (Netherlands))
The clinical syndrome of generalized, compensated glucocorticoid resistance is characterized by increased cortisol secretion without clinical evidence of hyper- or hypocortisolism, and manifestations of androgen and/or mineralocorticoid excess. This condition results from partial failure of the glucocorticoid receptor (GR) to modulate transcription of its target genes. The authors studied the molecular mechanisms of this syndrome in a Dutch kindred, whose affected members had hypercortisolism and approximately half of normal GRs, and whose proband was a young woman with manifestations of hyperandrogenism. Using the polymerase chain reaction to amplify and sequence each of the nine exons of the GR gene [alpha], along with their 5[prime]- and 3[prime]-flanking regions, the authors identified a 4-base deletion at the 3[prime]-boundary of exon 6 in one GR allele ([Delta][sub 4]), which removed a donor splice site in all three affected members studied. In contrast, the sequence of exon 6 in the two unaffected siblings was normal. A single nucleotide substitution causing an amino acid substitution in the amino terminal domain of the GR (asparagine to serine, codon 363) was also discovered in exon 2 of the other allele (G[sub 1220]) in the proband, in one of her affected brothers and in her unaffected sister. This deletion in the glucocorticoid receptor gene was associated with the expression of only one allele and a decrease of GR protein by 50% in affected members of this glucocorticoid resistant family. The mutation identified in exon 2 did not segregate with the disease and appears to be of no functional significance. The presence of the null allele was apparently compensated for by increased cortisol production at the expense of concurrent hyperandrogenism. 40 refs., 3 figs.
Digging into acceptor splice site prediction: an iterative feature selection approach
Gent, Universiteit
, Sven Degroeve, and Yves Van de Peer Department of Plant Systems Biology, Ghent University, Flanders will be irrelevant to the classification task, and will act as noise. This can degrade the performance. In this paper we will focus on the prediction of acceptor splice sites in the plant model species Arabidopsis
Myosin light-chain 1/3 gene alternative splicing: cis regulation is based upon a hierarchical compatibility between splice sites.
Gallego, M E; Nadal-Ginard, B
The mechanisms involved in the selective joining of appropriate 5' and 3' splice sites are still poorly understood in both constitutive and alternatively spliced genes. With two promoters associated with different exons, the myosin light-chain 1/3 gene generates two pre-mRNAs that also differ by the use of a pair of internal exons, 3 and 4, that are spliced in a mutually exclusive fashion. When the promoter upstream from exon 1 is used, only exon 4 is included. If the promoter upstream from exon 2 is used, only exon 3 is included. In an attempt to understand the molecular basis for the mutually exclusive behavior of these two exons and the basis of their specific selection, a number of minigene constructs containing exons 3 and 4 were tested in a variety of homologous or heterologous cis and trans environments. The results demonstrate that the mutually exclusive behavior of myosin light-chain exons 3 and 4 and selection between the two exons are cis regulated and are affected by the nature of the flanking sequences. Both exons competed for the common flanking 5' and 3' splice sites. Flanking exons were found that favored inclusion into mature mRNA of exon 3, exon 4, both, or neither, suggesting a specific cooperative interaction between certain 5' and 3' splice sites. Thus, alternative splicing of myosin light-chain 1/3 pre-mRNAs is regulated in cis by a hierarchy of compatibilities between pairs of 5' and 3' splice sites. Images PMID:2325649
A Recently Evolved Alternative Splice Site in the BRANCHED1a Gene Controls Potato Plant Architecture.
Nicolas, Michael; Rodr�guez-Buey, Mar�a Luisa; Franco-Zorrilla, Jos� Manuel; Cubas, Pilar
Amplification and diversification of transcriptional regulators that control development is a driving force of morphological evolution. A major source of�protein diversity is alternative splicing, which leads to the generation of different isoforms from a single gene. The mechanisms and timing of intron evolution nonetheless remain unclear, and the functions of alternative splicing-generated protein isoforms are rarely studied. In Solanum tuberosum, the BRANCHED1a (BRC1a) gene encodes a TCP transcription factor that controls lateral shoot outgrowth. Here, we report the recent evolution in Solanum of an alternative splice site in BRC1a that leads to the generation of two BRC1a protein isoforms with distinct C-terminal regions, BRC1a(Long) and BRC1a(Short), encoded by unspliced and spliced mRNA, respectively. The BRC1a(Long) C-terminal region has a strong activation domain, whereas that of BRC1a(S) lacks an activation domain and is predicted to form an amphipathic helix, the H domain, which prevents protein nuclear targeting. BRC1a(Short) is thus mainly cytoplasmic, while BRC1a(Long) is mainly nuclear. BRC1a(Long) functions as a transcriptional activator, whereas BRC1a(Short) appears to have no transcriptional activity. Moreover, BRC1a(Short) can heterodimerize with BRC1a(Long) and act as a dominant-negative factor; it increases BRC1a(Long) concentration in cytoplasm and reduces its transcriptional activity. This alternative splicing mechanism is regulated by hormones and external stimuli that control branching. The evolution of a new alternative splicing site and a novel protein domain in Solanum BRC1a led to a multi-level mechanism of post-transcriptional and post-translational BRC1a regulation that effectively modulates its branch suppressing activity in response to environmental and endogenous cues. PMID:26119747
Splicing mediates the activity of four putative cellular internal ribosome entry sites
Baranick, Brian T.; Lemp, Nathan A.; Nagashima, Jill; Hiraoka, Kei; Kasahara, Noriyuki; Logg, Christopher R.
A growing number of cellular mRNAs are thought to possess internal ribosome entry sites (IRESs), sequences that permit translation of a transcript independent of its 5? end and cap structure. Although dicistronic assays are the canonical method of testing sequences for IRES activity, they may produce false-positive results if unanticipated monocistronic RNAs arise from the dicistronic construct used. Using a dicistronic reporter system and a green fluorescent protein-tagged retrovirus to evaluate six previously reported cellular IRESs, we found that four contain 3? splice sites whose activity was required for apparent IRES function and which resulted in formation of monocistronic transcripts by splicing. Bioinformatic analysis revealed that the 3? splice sites identified in three of these putative IRESs are used in their native mRNAs and that the fourth is likely an artifactual sequence created during cDNA cloning. Our findings demonstrate a need for reexamination of other reported cellular IRESs by using careful RNA structural analysis to rule out splicing as the source of perceived IRES activity. PMID:18326627
Splicing mediates the activity of four putative cellular internal ribosome entry sites.
Baranick, Brian T; Lemp, Nathan A; Nagashima, Jill; Hiraoka, Kei; Kasahara, Noriyuki; Logg, Christopher R
A growing number of cellular mRNAs are thought to possess internal ribosome entry sites (IRESs), sequences that permit translation of a transcript independent of its 5' end and cap structure. Although dicistronic assays are the canonical method of testing sequences for IRES activity, they may produce false-positive results if unanticipated monocistronic RNAs arise from the dicistronic construct used. Using a dicistronic reporter system and a green fluorescent protein-tagged retrovirus to evaluate six previously reported cellular IRESs, we found that four contain 3' splice sites whose activity was required for apparent IRES function and which resulted in formation of monocistronic transcripts by splicing. Bioinformatic analysis revealed that the 3' splice sites identified in three of these putative IRESs are used in their native mRNAs and that the fourth is likely an artifactual sequence created during cDNA cloning. Our findings demonstrate a need for reexamination of other reported cellular IRESs by using careful RNA structural analysis to rule out splicing as the source of perceived IRES activity. PMID:18326627
Iliac crest bone graft donor site hernia: not so uncommon
Prabhu, Raghunath; Kumar, Nawin; Shenoy, Rajgopal
A 73-year-old man, who had undergone fracture fixation of humerus with LCP plating and bone grafting, presented again with a peri-implant fracture after 1?year. A repeat surgery was contemplated and replating was performed with tricortical bone graft harvested from the iliac crest. In the postoperative period the patient developed a tender swelling at the graft site with nausea and abdominal discomfort. It was managed conservatively thinking it to be a haematoma at the graft site. In the following 2?days his symptoms worsened. A CT abdomen showed the herniation of caecum from the bone graft donor site with obstruction. The patient was taken up for emergency surgical repair, the caecum was reduced and polypropylene mesh hernioplasty was performed. The patient recovered well without recurrence in the follow-up period. Iliac crest bone graft site hernia is not so uncommon and care has to be taken while harvesting. PMID:23761618
Exon 9 of the CFTR gene: splice site haplotypes and cystic fibrosis mutations.
D�rk, T; Fislage, R; Neumann, T; Wulf, B; T�mmler, B
The alternatively spliced exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene codes for the initial part of the amino-terminal nucleotide-binding fold of CFTR. A unique feature of the acceptor splice site preceding this exon is a variable length polymorphism within the polypyrimidine tract influencing the extent of exon 9 skipping in CFTR mRNA. We investigated this repeat for its relationship to CFTR mutations and intragenic markers on 200 chromosomes from German patients with cystic fibrosis (CF). Four frequent length variations were strongly associated with the four predominant haplotypes previously defined by intragenic marker dimorphisms. One of these alleles displayed absolute linkage disequilibrium to the major CF mutation delta F508. Other frequent CFTR mutations were linked to one particular splice site haplotype indicating that differential exon 9 skipping contributes little to the clinical heterogeneity among CF patients with an identical mutation. We also identified a novel missense mutation (V456F) and a novel nonsense mutation (Q414X) within the coding region of exon 9. The missense mutation V456F adjacent to Walker motif A was present in a pancreas-sufficient CF patient. In contrast, the pancreas-insufficient Q414X/delta F508 compound heterozygote suffered from a severe form of the disease, indicating that alternative splicing of exon 9 does not overcome the deleterious effect of a stop codon with this exon. PMID:7505767
Cross-kingdom patterns of alternative splicing and splice recognition
McGuire, Abigail Manson
Background: Variations in transcript splicing can reveal how eukaryotes recognize intronic splice sites. Retained introns (RIs) commonly appear when the intron definition (ID) mechanism of splice site recognition inconsistently ...
Active site modification of aldose reductase by nitric oxide donors.
Chandra, A; Srivastava, S; Petrash, J M; Bhatnagar, A; Srivastava, S K
Nitric oxide (NO) donors sodium nitrosoprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosydnonemine (SIN-1) caused a time- and concentration-dependent loss of catalytic activity of recombinant human placental aldose reductase. Modification of the enzyme was prevented by NADPH and NADP and reversed partially by dithiothreitol (DTT) and sodium borohydride. The protection by NADPH was lost in the presence of both substrates (NADPH and glyceraldehyde), indicating that the enzyme becomes sensitive to inhibition by SNP during catalysis. Site-directed mutant form of the enzyme, in which active site cys-298 was substituted with serine (C298S) was not inactivated by NO donors, whereas, ARC80S and ARC303 were as sensitive as the wild type enzyme, indicating that inactivation of aldose reductase is due to modification of the active site at cys298. These results suggest that NO may be an endogenous regulator of aldose reductase, and consequently the polyol pathway of glucose metabolism; which has been implicated in the pathogenesis of secondary diabetic complications. PMID:9357961
Achieving ideal donor site aesthetics with autologous breast reconstruction
The appearance of the donor site following breast reconstruction with abdominal flaps has become an important topic for study. Given the variety of flaps that are derived from the abdomen, decisions are often based on how much muscle and fascia will be harvested. Comparisons between muscle sparing and non-muscle sparing techniques have been performed with outcomes related to function and contour. Closure techniques will vary and include primary fascial closure, mesh reinforcement and additional fascial plication all of which can produce natural and sometimes improved abdominal contours. Proper patient selection however is important. This manuscript will describe various techniques in order to achieve ideal abdominal contour following autologous reconstruction. PMID:26005646
A novel splice site mutation in the WAS gene causes Wiskott-Aldrich syndrome in two siblings of a Saudi family.
Abu-Amero, Khaled K; Owaidah, Tarek M; Al Jefri, Abduallah; Al-Ghonaium, Abdulaziz; Fawaz, Ibrahim M; Al-Hamed, Mohammed H
We report here on a Saudi family with two affected males with Wiskott-Aldrich syndrome (WAS), which includes mild to moderate bleeding and a low platelet count. A novel splice donor-site mutation (811 + 5 G <-- C) in intron 8 of the WAS gene (Genbank accession number NM_000377) was detected in a hemizygous status in both index cases, heterozygous in their mother and absent in the father. RNA from both index cases was transcribed and amplified with primers complementary to sequences in exons 7 and 10. A reverse transcription-polymerase chain reaction (RT-PCR) product of 688 bp (approximately 82%) was produced in addition to the normal RT-PCR product of 485 bp (approximately 18%). cDNA sequence analysis reveals an inclusion of full intron 8 sequence in the final transcript. The resultant protein is predicted to have 68 missense codons and a pre-mature stop codon at amino acid 260. This novel splice donor-site mutation was not detected in 80 normal controls (56 females and 24 males) from the same ethnic background as the index cases. Since no other mutation was detected in the WAS gene and the patients have classical symptoms of WAS, we concluded that it is highly likely that this novel mutation is responsible for the phenotype observed in these patients. PMID:15389128
Principles of 3 splice site selection and alternative splicing for an unusual group II intron from Bacillus anthracis
Zimmerly, Steven
Bacillus anthracis AARON R. ROBART,1 NANCY KRISTINE MONTGOMERY,2 KIMOTHY L. SMITH,2 and STEVEN ZIMMERLY1 1 Bacillus anthracis. One intron (B.a.I1) splices poorly in vitro despite having typical structural motifs
Spliced transcripts of human cytomegalovirus.
Rawlinson, W D; Barrell, B G
The availability of the human cytomegalovirus (HCMV) genomic sequence has resulted in more extensive knowledge of the overall coding capacity of the virus. Using polymerase chain reaction and rapid sequencing techniques, we have studied the splicing of mRNAs from a number of the predicted open reading frames (ORFs). Splicing was found between the UL122(IE2) ORF present within major immediate-early (MIE) region 2 and the downstream ORF (UL118) predicted to encode an incomplete glycoprotein. This locates the IE2 3' donor site and provides evidence of a link between the MIE region and downstream ORFs. The downstream UL119-UL118-UL115 ORFs also undergo differential splicing, further increasing the known complexity of this region of the genome. A detailed map of the differential splicing within the region encoding the MIE ORF is presented. Also described are several previously unidentified spliced ORFs found in the long repeats and long unique regions, including one encoding a transcript with a large (4-kb) intron. The results show that spliced transcripts are encoded from throughout the genome at immediate-early, early, and late times postinfection. Images PMID:7688825
BAP1 Missense Mutation c.2054 A>T (p.E685V) Completely Disrupts Normal Splicing through Creation of a Novel 5� Splice Site in a Human Mesothelioma Cell Line
Morrison, Arianne; Chekaluk, Yvonne; Bacares, Ruben; Ladanyi, Marc; Zhang, Liying
BAP1 is a tumor suppressor gene that is lost or deleted in diverse cancers, including uveal mela�noma, malignant pleural mesothelioma (MPM), clear cell renal carcinoma, and cholangiocarcinoma. Recently, BAP1 germline mutations have been reported in families with combinations of these same cancers. A particular challenge for mutation screening is the classification of non-truncating BAP1 sequence variants because it is not known whether these subtle changes can affect the protein function sufficiently to predispose to cancer development. Here we report mRNA splicing analysis on a homozygous substitution mutation, BAP1 c. 2054 A&T (p.Glu685Val), identified in an MPM cell line derived from a mesothelioma patient. The mutation occurred at the 3rd nucleotide from the 3� end of exon 16. RT-PCR, cloning and subsequent sequencing revealed several aberrant splicing products not observed in the controls: 1) a 4 bp deletion at the end of exon 16 in all clones derived from the major splicing product. The BAP1 c. 2054 A&T mutation introduced a new 5� splice site (GU), which resulted in the deletion of 4 base pairs and presumably protein truncation; 2) a variety of alternative splicing products that led to retention of different introns: introns 14�16; introns 15�16; intron 14 and intron 16; 3) partial intron 14 and 15 retentions caused by activation of alternative 3� splice acceptor sites (AG) in the introns. Taken together, we were unable to detect any correctly spliced mRNA transcripts in this cell line. These results suggest that aberrant splicing caused by this mutation is quite efficient as it completely abolishes normal splicing through creation of a novel 5� splice site and activation of cryptic splice sites. These data support the conclusion that BAP1 c.2054 A&T (p.E685V) variant is a pathogenic mutation and contributes to MPM through disruption of normal splicing. PMID:25830670
Biased exon/intron distribution of cryptic and de novo 3? splice sites
Kr�lovi?ov�, Jana; Christensen, Mikkel B.; Vo?echovsk�, Igor
We compiled sequences of previously published aberrant 3? splice sites (3?ss) that were generated by mutations in human disease genes. Cryptic 3?ss, defined here as those resulting from a mutation of the 3?YAG consensus, were more frequent in exons than in introns. They clustered in ?20 nt region adjacent to authentic 3?ss, suggesting that their under-representation in introns is due to a depletion of AG dinucleotides in the polypyrimidine tract (PPT). In contrast, most aberrant 3?ss that were induced by mutations outside the 3?YAG consensus (designated �de novo�) were in introns. The activation of intronic de novo 3?ss was largely due to AG-creating mutations in the PPT. In contrast, exonic de novo 3?ss were more often induced by mutations improving the PPT, branchpoint sequence (BPS) or distant auxiliary signals, rather than by direct AG creation. The Shapiro�Senapathy matrix scores had a good prognostic value for cryptic, but not de novo 3?ss. Finally, AG-creating mutations in the PPT that produced aberrant 3?ss upstream of the predicted BPS in vivo shared a similar �BPS-new AG� distance. Reduction of this distance and/or the strength of the new AG PPT in splicing reporter pre-mRNAs improved utilization of authentic 3?ss, suggesting that AG-creating mutations that are located closer to the BPS and are preceded by weaker PPT may result in less severe splicing defects. PMID:16141195
The 3? Splice Site of Influenza A Segment 7 mRNA Can Exist in Two Conformations: A Pseudoknot and a Hairpin
Walter N. Moss; Lumbini I. Dela-Moss; Elzbieta Kierzek; Ryszard Kierzek; Salvatore F. Priore; Douglas H. Turner
The 3? splice site of influenza A segment 7 is used to produce mRNA for the M2 ion-channel protein, which is critical to the formation of viable influenza virions. Native gel analysis, enzymatic\\/chemical structure probing, and oligonucleotide binding studies of a 63 nt fragment, containing the 3? splice site, key residues of an SF2\\/ASF splicing factor binding site, and a
Dysferlin rescue by spliceosome-mediated pre-mRNA trans-splicing targeting introns harbouring weakly defined 3' splice sites.
Philippi, Susanne; Lorain, St�phanie; Beley, Cyriaque; Peccate, C�cile; Pr�cigout, Guillaume; Spuler, Simone; Garcia, Luis
The modification of the pre-mRNA cis-splicing process employing a pre-mRNA trans-splicing molecule (PTM) is an attractive strategy for the in situ correction of genes whose careful transcription regulation and full-length expression is determinative for protein function, as it is the case for the dysferlin (DYSF, Dysf) gene. Loss-of-function mutations of DYSF result in different types of muscular dystrophy mainly manifesting as limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi muscular dystrophy 1 (MMD1). We established a 3' replacement strategy for mutated DYSF pre-mRNAs induced by spliceosome-mediated pre-mRNA trans-splicing (SmaRT) by the use of a PTM. In contrast to previously established SmaRT strategies, we particularly focused on the identification of a suitable pre-mRNA target intron other than the optimization of the PTM design. By targeting DYSF pre-mRNA introns harbouring differentially defined 3' splice sites (3' SS), we found that target introns encoding weakly defined 3' SSs were trans-spliced successfully in vitro in human LGMD2B myoblasts as well as in vivo in skeletal muscle of wild-type and Dysf(-/-) mice. For the first time, we demonstrate rescue of Dysf protein by SmaRT in vivo. Moreover, we identified concordant qualities among the successfully targeted Dysf introns and targeted endogenous introns in previously reported SmaRT approaches that might facilitate a selective choice of target introns in future SmaRT strategies. PMID:25904108
Rational selection of flaps from the abdomen in breast reconstruction to reduce donor site morbidity
Z. M. Arnez; U. Khan; D. Pogorelec; F. Planinsek
Reconstruction of the female breast following mastectomy has become commonplace. The number of donor sites have increased as the quest both for improving reconstruction and reducing morbidity continues. There are a number of donor sites which resemble breast tissue in terms of skin texture, suppleness and colour. The �gold standard� for transfer in breast reconstruction, however, is the lower abdominal
Preventing seroma in the latissimus dorsi flap donor site with fibrin sealant.
Weinrach, Jonathan C; Cronin, Ernest D; Smith, Bruce K; Collins, Donald R; Cohen, Benjamin E
Donor site seroma continues to be the most common complication of latissimus dorsi flap reconstruction. Numerous preventive methods and treatments have been described. The use of fibrin sealant in the donor site before closure may be an effective means of seroma prevention. The authors evaluated the efficacy of fibrin sealant in conjunction with closed suction drainage in a series of 17 latissimus flap donor sites. They also determined their approximate institutional seroma rate with a retrospective chart review of 20 donor sites. The fibrin sealant patients had a seroma rate of 11.8% compared with a rate of 35% among the untreated patients (P = 0.047). This compares favorably with seroma rates as high as 79% described in the literature. In conclusion, the use of fibrin sealant in the latissimus flap donor site appears to be effective in preventing seroma. PMID:15211191
Altered PLP1 splicing causes hypomyelination of early myelinating structures
Kevelam, Sietske H; Taube, Jennifer R; van Spaendonk, Rosalina M L; Bertini, Enrico; Sperle, Karen; Tarnopolsky, Mark; Tonduti, Davide; Valente, Enza Maria; Travaglini, Lorena; Sistermans, Erik A; Bernard, Genevi�ve; Catsman-Berrevoets, Coriene E; van Karnebeek, Clara D M; �stergaard, John R; Friederich, Richard L; Fawzi Elsaid, Mahmoud; Schieving, Jolanda H; Tarailo-Graovac, Maja; Orcesi, Simona; Steenweg, Marjan E; van Berkel, Carola G M; Waisfisz, Quinten; Abbink, Truus E M; van der Knaap, Marjo S; Hobson, Grace M; Wolf, Nicole I
Objective The objective of this study was to investigate the genetic etiology of the X-linked disorder �Hypomyelination of Early Myelinating Structures� (HEMS). Methods We included 16 patients from 10 families diagnosed with HEMS by brain MRI criteria. Exome sequencing was used to search for causal mutations. In silico analysis of effects of the mutations on splicing and RNA folding was performed. In vitro gene splicing was examined in RNA from patients� fibroblasts and an immortalized immature oligodendrocyte cell line after transfection with mutant minigene splicing constructs. Results All patients had unusual hemizygous mutations of PLP1 located in exon 3B (one deletion, one missense and two silent), which is spliced out in isoform DM20, or in intron 3 (five mutations). The deletion led to truncation of PLP1, but not DM20. Four mutations were predicted to affect PLP1/DM20 alternative splicing by creating exonic splicing silencer motifs or new splice donor sites or by affecting the local RNA structure of the PLP1 splice donor site. Four deep intronic mutations were predicted to destabilize a long-distance interaction structure in the secondary PLP1 RNA fragment involved in regulating PLP1/DM20 alternative splicing. Splicing studies in fibroblasts and transfected cells confirmed a decreased PLP1/DM20 ratio. Interpretation Brain structures that normally myelinate early are poorly myelinated in HEMS, while they are the best myelinated structures in Pelizaeus�Merzbacher disease, also caused by PLP1 alterations. Our data extend the phenotypic spectrum of PLP1-related disorders indicating that normal PLP1/DM20 alternative splicing is essential for early myelination and support the need to include intron 3 in diagnostic sequencing.
Squamous cell carcinoma of the pectoralis major myocutaneous flap donor site.
Mohan, A Mathan; Balaguhan, B; Krishna, Vinod; Nagarjuna, Muralidhara
The pectoralis major myocutaneous flap is considered a workhorse flap in the reconstruction of head and neck defects after cancer ablative surgeries and remains one of the most widely used reconstructive options. Complications at the donor site after the use of this flap, although rare, do occur and are usually restricted to minor infections, hematoma, and seroma formation. Metastasis to the flap donor site is a rare complication with limited documentation. Metastasis at the donor site usually follows local recurrence at the primary site, supporting the probable hypothesis of re-establishment of lymphatic drainage to the primary site by the flap pedicle. Tumor implantation, although a probable cause for metastasis at the donor site, cannot be confidently distinguished from other mechanisms, such as hematogenous spread or lymphogenous metastasis. This report describes a case that supports a seeding or tumor implantation mechanism of metastasis exclusively. PMID:24560174
Human papillomavirus type 16 E2 and E6 are RNA-binding proteins and inhibit in vitro splicing of pre-mRNAs with suboptimal splice sites
Bodaghi, Sohrab; Jia Rong [HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (United States); Zheng Zhiming [HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (United States)], E-mail: email@example.com
Human papillomavirus type 16 (HPV16) genome expresses six regulatory proteins (E1, E2, E4, E5, E6, and E7) which regulate viral DNA replication, gene expression, and cell function. We expressed HPV16 E2, E4, E6, and E7 from bacteria as GST fusion proteins and examined their possible functions in RNA splicing. Both HPV16 E2, a viral transactivator protein, and E6, a viral oncoprotein, inhibited splicing of pre-mRNAs containing an intron with suboptimal splice sites, whereas HPV5 E2 did not. The N-terminal half and the hinge region of HPV16 E2 as well as the N-terminal and central portions of HPV16 E6 are responsible for the suppression. HPV16 E2 interacts with pre-mRNAs through its C-terminal DNA-binding domain. HPV16 E6 binds pre-mRNAs via nuclear localization signal (NLS3) in its C-terminal half. Low-risk HPV6 E6, a cytoplasmic protein, does not bind RNA. Notably, both HPV16 E2 and E6 selectively bind to the intron region of pre-mRNAs and interact with a subset of cellular SR proteins. Together, these findings suggest that HPV16 E2 and E6 are RNA binding proteins and might play roles in posttranscriptional regulation during virus infection.
Germinal HPRT splice donor site mutation results in multiple RNA splicing products in T-lymphocyte cultures
Hunter, T.C.; Albertini, R.J.; O`Neill, J.P. [Univ. of Vermont Genetics Lab., Burlington, VT (United States)] [and others
Fanconi anemia (FA) is an autosomal recessive disease characterized by birth defects, progressive bone marrow failure and increased risk for leukemia. FA cells display chromosome breakage and increased cell killing in response to DNA crosslinking agents. At least 5 genes have been defined by cell complementation studies, but only one of these, FAC has been cloned to date. Efforts to map and isolate new FA genes by functional complementation have been hampered by the lack of immortalized FA fibroblast cell lines. Here we report the use of a novel immortalization strategy to create 4 new immortalized FA fibroblast lines, including one from the rare complementation group D. 16 refs., 3 tabs.
Cross-kingdom patterns of alternative splicing and splice recognition
Abigail M McGuire; Matthew D Pearson; Daniel E Neafsey; James E Galagan
Background��Variations in transcript splicing can reveal how eukaryotes recognize intronic splice sites. Retained introns (RIs) commonly\\u000a appear when the intron definition (ID) mechanism of splice site recognition inconsistently identifies intron-exon boundaries,\\u000a and cassette exons (CEs) are often caused by variable recognition of splice junctions by the exon definition (ED) mechanism.\\u000a We have performed a comprehensive survey of alternative splicing across
Probing the Role of a Secondary Structure Element at the 5�- and 3�Splice Sites in Group I Intron Self-Splicing:� The Tetrahymena L-16 Sca I Ribozyme Reveals a New Role of the G�U Pair in Self-Splicing �
Katrin Karbstein; Jihee Lee; Daniel Herschlag
Several ribozyme constructs have been used to dissect aspects of the group I self-splicing reaction. The Tetrahymena L-21 ScaI ribozyme, the best studied of these intron analogues, catalyzes a reaction analogous to the first step of self-splicing, in which a 5 '-splice site analogue (S) and guanosine (G) are converted into a 5'-exon analogue (P) and GA. This ribozyme preserves
Donor site morbidity of the fasciocutaneous radial forearm flap: what does the patient really bother?
de Witt, Christien A.; Verdonck-de Leeuw, Irma M.; Quak, Jasper J.; Leemans, C. Ren�
The objective of this study was the evaluation of donor site morbidity in head and neck cancer patients after reconstruction using a free vascularized radial forearm flap with emphasis on subjective complaints. Fifty patients who underwent at least 6�months before a reconstruction using a free vascularized radial forearm flap were asked to fill out two questionnaires regarding cosmetics and sensibility and forearm disabilities. Furthermore, a function test including movement extensions (flexion�extension, ulnar�radial deviation and pronation�supination), strength (pinch and grip) and temperature (digiti I and V) of the donor and non-donor site were measured and compared. Thirty-five percent of the patients reported no complaints regarding cosmetics and sensibility and 75% mentioned no forearm disabilities. There was no difference in movement extensions, temperature and grip strength between donor and non-donor sites. The difference in pinch strength appeared to be significant (p�<�0.001). The total score of the questionnaire on forearm disabilities correlated significantly with extension, pronation and grip strength of the donor arm. Donor site morbidity of the radial forearm flap measured by objective functional tests was limited but subjective self-ratings revealed complaints regarding cosmestics and sensibility and to a lesser extent regarding forearm disability. The present data may be used for solid patient counselling. PMID:17323087
Alteration of splice site selection in the LMNA gene and inhibition of progerin production via AMPK activation.
Finley, Jahahreeh
Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by an accelerated aging phenotype and an average life span of 13years. Patients typically exhibit extensive pathophysiological vascular alterations, eventually resulting in death from stroke or myocardial infarction. A silent point mutation at position 1824 (C1824T) of the LMNA gene, generating a truncated form of lamin A (progerin), has been shown to be the cause of most cases of HGPS. Interestingly, this mutation induces the use of an internal 5' cryptic splice site within exon 11 of the LMNA pre-mRNA, leading to the generation of progerin via aberrant alternative splicing. The serine-arginine rich splicing factor 1 (SRSF1 or ASF/SF2) has been shown to function as an oncoprotein and is upregulated in many cancers and other age-related disorders. Indeed, SRSF1 inhibition results in a splicing ratio in the LMNA pre-mRNA favoring lamin A production over that of progerin. It is our hypothesis that activation of AMP-activated protein kinase (AMPK), a master regulator of cellular metabolism, may lead to a reduction in SRSF1 and thus a decrease in the use of the LMNA 5' cryptic splice site in exon 11 through upregulation of p32, a splicing factor-associated protein and putative mitochondrial chaperone that has been shown to inhibit SRSF1 and enhance mitochondrial DNA (mtDNA) replication and oxidative phosphorylation. AMPK activation by currently available compounds such as metformin, resveratrol, and berberine may thus have wide-ranging implications for disorders associated with increased production and accumulation of progerin. PMID:25216752
A canonical splice site mutation in GIPC3 causes sensorineural hearing loss in a large Pakistani family.
Siddiqi, Saima; Ismail, Muhammad; Oostrik, Jaap; Munawar, Saba; Mansoor, Atika; Kremer, Hannie; Qamar, Raheel; Schraders, Margit
With homozygosity mapping we have identified two large homozygous regions on chromosome 3q13.11-q13.31 and chromosome 19p13.3-q31.32 in a large Pakistani family suffering from autosomal recessive nonsyndromic hearing impairment (arNSHI). The region on chromosome 19 overlaps with the previously described deafness loci DFNB15, DFNB72 and DFNB95. Mutations in GIPC3 have been shown to underlie the nonsyndromic hearing impairment linked to these loci. Sequence analysis of all exons and exon-intron boundaries of GIPC3 revealed a homozygous canonical splice site mutation, c.226-1G>T, in GIPC3. This is the first mutation described in GIPC3 that affects splicing. The c.226-1G>T mutation is located in the acceptor splice site of intron 1 and is predicted to affect the normal splicing of exon 2. With a minigene assay it was shown to result in the use of an alternative acceptor site in exon 2, resulting in a frameshift and a premature stop codon. This study expands the mutational spectrum of GIPC3 in arNSHI. PMID:25296581
Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR)
Solomon, Oz; Oren, Shirley; Safran, Michal; Deshet-Unger, Naamit; Akiva, Pinchas; Jacob-Hirsch, Jasmine; Cesarkas, Karen; Kabesa, Reut; Amariglio, Ninette; Unger, Ron; Rechavi, Gideon; Eyal, Eran
Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery. PMID:23474544
The missing puzzle piece: splicing mutations
Lewandowska, Marzena A
Proper gene splicing is highly dependent on the correct recognition of exons. Among the elements allowing this process are the �cis� (conserved sequences) and �trans� (snRNP, splicing factors) elements. Splicing mutations are related with a number of genetic disorders and usually induce exon skipping, form new exon/intron boundaries or activate new cryptic exons as a result of alterations at donor/acceptor sites. They constitute more than 9% of the currently published mutations, but this value is highly underestimated as many of the potential mutations are located in the �cis� elements and should be confirmed experimentally. The most commonly detected splicing mutations are located at donor (5�) and acceptor (3�) sites. Mutations at the branch point are rare (only over a dozen are known to date), and are mostly searched and detected when no alteration has been detected in the sequenced exons and UTRs. Polypyrimidine tract mutations are equally rare. High throughput technologies, as well as traditional Sanger sequencing, allow detection of many changes in intronic sequences and intron/exon boundaries. However, the assessment whether a mutation affects exon recognition and results in a genetic disorder has to be conducted using molecular biology methods: in vitro transcription of the sequence of interest cloned into a plasmid, with and without alterations, or mutation analysis via a hybrid minigene system. Even though microarrays and new generation sequencing methods pose difficulties in detecting novel branch point mutations, these tools seem appropriate to expand the mutation detection panel especially for diagnostic purposes. PMID:24294354
Intensive insulin treatment increases donor site wound protein synthesis in burn patients
Tuvdendorj, Demidmaa; Zhang, Xiao-Jun; Chinkes, David L.; Aarsland, Asle; Kulp, Gabriela A.; Jeschke, Marc G.; Herndon, David N.
Background In the treatment of burns, patients� own skin is the preferred material to cover burn wounds, resulting in the need to create a donor site wound. Enhancement of healing of the donor site wound would be beneficial in burn patients. Insulin, an anabolic agent, is routinely used to treat hyperglycemia after injury. We investigated whether intensive insulin treatment (INS) increases fractional synthesis rate (FSR) of the donor site wound protein and decreases the length of hospitalization normalized for total body surface area burned (LOS/TBSA). Methods FSR of the donor site wound protein was measured in pediatric patients randomized to control (CNT) (n = 13) and INS (n = 10) treatments. Depending on the postoperative day when the tracer study was done studies were divided into �Early� (days < 5) and �Late� (days >=5) periods. Results FSR of the donor site wound protein was greater in the INS group at the �Early� period of wound healing (CNT vs. INS, 8.2�3.8 vs. 13.1�6.9 %/day, p: < 0.05); but not at the �Late� (CNT vs. INS, 19.7�4.6 vs. 16.6�4.0 %/day, p > 0.05). Despite these differences LOS/TBSA was not decreased in the INS group. Correlation analyses demonstrated that independently of the treatment regimen FSR positively correlated (p < 0.05) with time post creation of the donor site and negatively correlated (p < 0.05) with LOS/TBSA. Conclusions Insulin treatment increased FSR of the donor site wound protein in the early period of wound healing; FSR correlated with LOS/TBSA independently of the treatment regimen. PMID:21236451
Widespread and subtle: alternative splicing at short-distance tandem sites
Will, Sebastian
], and the regulation of alternative splicing is important for diverse biological processes. For example, the brain-specific species. It results in subtle changes in the transcripts and often in the encoded proteins. Several of these tandem splice events contribute to the repertoire of functionally different proteins, whereas many
The Choice of Alternative 5' Splice Sites in Influenza Virus M1 mRNA is Regulated by the Viral Polymerase Complex
NASA Astrophysics Data System (ADS)
Shih, Shin-Ru; Nemeroff, Martin E.; Krug, Robert M.
The influenza virus M1 mRNA has two alternative 5' splice sites: a distal 5' splice site producing mRNA_3 that has the coding potential for 9 amino acids and a proximal 5' splice site producing M2 mRNA encoding the essential M2 ion-channel protein. Only mRNA_3 was made in uninfected cells transfected with DNA expressing M1 mRNA. Similarly, using nuclear extracts from uninfected cells, in vitro splicing of M1 mRNA yielded only mRNA_3. Only when the mRNA_3 5' splice site was inactivated by mutation was M2 mRNA made in uninfected cells and in uninfected cell extracts. In influenza virus-infected cells, M2 mRNA was made, but only after a delay, suggesting that newly synthesized viral gene product(s) were needed to activate the M2 5' splice site. We present strong evidence that these gene products are the complex of the three polymerase proteins, the same complex that functions in the transcription and replication of the viral genome. Gel shift experiments showed that the viral polymerase complex bound to the 5' end of the viral M1 mRNA in a sequence-specific and cap-dependent manner. During in vitro splicing catalyzed by uninfected cell extracts, the binding of the viral polymerase complex blocked the mRNA_3 5' splice site, resulting in the switch to the M2 mRNA 5' splice site and the production of M2 mRNA.
Length changes in the joining segment between domains 5 and 6 of a group II intron inhibit self-splicing and alter 3' splice site selection.
Boulanger, S C; Faix, P H; Yang, H; Zhuo, J; Franzen, J S; Peebles, C L; Perlman, P S
Domain 5 (D5) and domain 6 (D6) are adjacent folded hairpin substructures of self-splicing group II introns that appear to interact within the active ribozyme. Here we describe the effects of changing the length of the 3-nucleotide segment joining D5 to D6 [called J(56)3] on the splicing reactions of intron 5 gamma of the COXI gene of yeast mitochondrial DNA. Shortened variants J(56)0 and J(56)1 were defective in vitro for branching, and the second splicing step was performed inefficiently and inaccurately. The lengthened variant J(56)5 had a milder defect-splicing occurred at a reduced rate but with correct branching and a mostly accurate 3' splice junction choice. Yeast mitochondria were transformed with the J(56)5 allele, and the resulting yeast strain was respiration deficient because of ineffective aI5 gamma splicing. Respiration-competent revertants were recovered, and in one type a single joiner nucleotide was deleted while in the other type a nucleotide of D6 was deleted. Although these revertants still showed partial splicing blocks in vivo and in vitro, including a substantial defect in the second step of splicing, both spliced accurately in vivo. These results establish that a 3-nucleotide J(56) is optimal for this intron, especially for the accuracy of 3' splice junction selection, and indicate that D5 and D6 are probably not coaxially stacked. PMID:8816503
Comparative Clinical Study of Bactigras and Telfa AMD for Skin Graft Donor-Site Dressing
Muangman, Pornprom; Nitimonton, Sooksan; Aramwit, Pornanong
The Bactigras� paraffin tulle coated with chlorhexidine is normally used for the treatment of donor-site wounds in burn patients who received split-thickness skin grafts in several centers. It has some disadvantages, such as adhesion to wound surfaces and pain from the irritation caused by this dressing. The Telfa AMD�, a non-adherent wound dressing which consists of absorbent cotton fibers impregnated with polyhexamethylene biguanide enclosed in a sleeve of thermoplastic polymers, is a new option for donor-site wound care which causes less adherence to the wound. The purpose of this study was to compare clinical efficacy of these two dressings for the management of donor-site wounds. Thirty-two patients who received split-thickness skin grafts by donor site harvesting from the thigh were enrolled in this study and randomized into two groups receiving either the Bactigras� or the Telfa AMD� wound treatment. Re-epithelialization, pain, infection and cost-effectiveness analyses were compared between both groups. The results showed that there was no significant difference in age, area of donor sites or length of hospital stays between the groups (p > 0.05). However, the day of re-epithelialization (?90%) was significantly shorter in patients treated with the Telfa AMD� compared to the Bactigras� group (14.00 � 3.05 vs. 9.25 � 1.88 days for Bactigras� and Telfa AMD� groups, respectively, p < 0.001). The average pain score was also significantly lower in the Telfa AMD� group (1.57 � 0.55 vs. 4.70 � 1.16, p < 0.001). There was no difference in the cost of treatment between the groups (4.64 � 1.97 vs. 5.72 � 2.54 USD, p = 0.19). This study indicated that the Telfa AMD� was an effective dressing for the treatment of donor-site wounds. PMID:21954342
Directing alternative splicing: cast and scenarios
Benoit Chabot
Recent progress in the study of alternative RNA splicing indicates that the interaction of RNA-binding proteins with specific target elements modulates splice site recognition and spliceosome assembly. The identity of splicing signals, the presence of modulating elements and differences in the distribution of RNA-binding proteins are key determinants involved in the tissue-specific regulation of splice site selection.
Lysyl oxidase-like 4 is alternatively spliced in an anatomic site-specific manner in tumors involving the serosal cavities.
Sebban, Shulamit; Davidson, Ben; Reich, Reuven
Lysyl oxidase-like enzymes (LOXL) are expressed in various cancers. We analyzed the expression of LOXL2, LOXL3, and LOXL4 in cancers involving the serosal cavities-breast carcinoma, ovarian carcinoma, and malignant mesothelioma using reverse-transcriptase polymerase chain reaction. We discovered two new alternative splice variants of LOXL4. The spliced segments were exon 9 (splice variant 1) or both exons 8 and 9 (splice variant 2). In ovarian carcinoma, splice variant 1 was significantly elevated in effusions compared to solid lesions (p < 0.001). Splice variant 2 appeared only in effusions. In breast carcinoma, LOXL4 was expressed only in the effusion samples. In malignant mesothelioma, LOXL4 and its splice variants were expressed at all sites. Breast carcinoma effusions showed significantly higher LOXL2 (p = 0.003) and lower LOXL3 (p < 0.001) expression compared to primary carcinomas. Our data show differences in LOXL messenger RNA expression as a function of anatomic site and tumor type in cancers affecting the serosal cavities. PMID:19015874
Apparent Defect in Yeast Bud-Site Selection Due to a Specific Failure to Splice the Pre-mRNA of a Regulator of
Pringle, John
Apparent Defect in Yeast Bud-Site Selection Due to a Specific Failure to Splice the Pre The yeast Saccharomyces cerevisiae normally selects bud sites (and hence axes of cell polarization) in one. Although many of the proteins involved in bud-site selection are known, it is likely that others remain
Transcriptome sequencing reveals potential mechanism of cryptic 3' splice site selection in SF3B1-mutated cancers.
DeBoever, Christopher; Ghia, Emanuela M; Shepard, Peter J; Rassenti, Laura; Barrett, Christian L; Jepsen, Kristen; Jamieson, Catriona H M; Carson, Dennis; Kipps, Thomas J; Frazer, Kelly A
Mutations in the splicing factor SF3B1 are found in several cancer types and have been associated with various splicing defects. Using transcriptome sequencing data from chronic lymphocytic leukemia, breast cancer and uveal melanoma tumor samples, we show that hundreds of cryptic 3' splice sites (3'SSs) are used in cancers with SF3B1 mutations. We define the necessary sequence context for the observed cryptic 3' SSs and propose that cryptic 3'SS selection is a result of SF3B1 mutations causing a shift in the sterically protected region downstream of the branch point. While most cryptic 3'SSs are present at low frequency (<10%) relative to nearby canonical 3'SSs, we identified ten genes that preferred out-of-frame cryptic 3'SSs. We show that cancers with mutations in the SF3B1 HEAT 5-9 repeats use cryptic 3'SSs downstream of the branch point and provide both a mechanistic model consistent with published experimental data and affected targets that will guide further research into the oncogenic effects of SF3B1 mutation. PMID:25768983
Characterization of novel SLC6A8 variants with the use of splice-site analysis tools and implementation of a newly developed LOVD database
Betsalel, Ofir T; Rosenberg, Efraim H; Almeida, Ligia S; Kleefstra, Tjitske; Schwartz, Charles E; Valayannopoulos, Vassili; Abdul-Rahman, Omar; Poplawski, Nicola; Vilarinho, Laura; Wolf, Philipp; den Dunnen, Johan T; Jakobs, Cornelis; Salomons, Gajja S
The X-linked creatine transporter defect is caused by mutations in the SLC6A8 gene. Until now, 66 synonymous and intronic variants in SLC6A8 were detected in our laboratory. To gain more insight in the effect of the detected variants, we applied five free web-based splice-site analysis tools to 25 published variants that were stratified as (non-)disease causing. All were correctly predicted to have no effect (n=18) or to cause erroneous splicing (n=7), with the exception of a pathogenic de novo 24?bp intronic deletion. Second, 41 unclassified variants, including 28 novel, were subjected to analysis by these tools. At least four splice-site analysis tools predicted that three of the variants would affect splicing as the mutations disrupted the canonical splice site. Urinary creatine/creatinine and brain MRS confirmed creatine transporter deficiency in five patients (four families), including one female. Another variant was predicted to moderately affect splicing by all five tools. However, transient transfection of a minigene containing the variant in a partial SLC6A8 segment showed no splicing errors, and thus was finally classified as non-disease causing. This study shows that splice tools are useful for the characterization of the majority of variants, but also illustrates that the actual effect can be misclassified in rare occasions. Therefore, further laboratory studies should be considered before final conclusions on the disease-causing nature are drawn. To provide an accessible database, the 109 currently known SLC6A8 variants, including 35 novel ones, are included in a newly developed LOVD DNA variation database. PMID:20717164
Modulation of SMN pre-mRNA splicing by inhibition of alternative 3' splice site pairing Sharlene R. Lim and Klemens J. Hertel*
Hertel, Klemens J.
(SMA), Survival Motor Neuron (SMN), antisense oligonucleotide Running Title: SMN Differential Splicing inclusion. These results suggest that antisense oligonucleotides could be used as a therapeutic strategy but more importantly, increase the efficiency of the competing exon 7 skipping pathway. Antisense
In vitro analysis of splice site mutations in the CLCN1 gene using the minigene assay.
Ulzi, Gianna; Sansone, Valeria A; Magri, Francesca; Corti, Stefania; Bresolin, Nereo; Comi, Giacomo P; Lucchiari, Sabrina
Mutations in the chloride channel gene CLCN1 cause the allelic disorders Thomsen (dominant) and Becker (recessive) myotonia congenita (MC). The encoded protein, ClC-1, is the primary channel that mediates chloride (Cl-) conductance in skeletal muscle. Mutations in CLCN1 lower the channel's threshold voltage, leading to spontaneous action potentials that are not coupled to neuromuscular transmission and resulting in myotonia. Over 120 mutations in CLCN1 have been described, 10% of which are splicing defects. Biological specimens suitable for RNA extraction are not always available, but obtaining genomic DNA for analysis is easy and non-invasive. This is the first study to evaluate the pathogenic potential of novel splicing mutations using the minigene approach, which is based on genomic DNA analysis. Splicing mutations accounted for 23% of all pathogenic variants in our cohort of MC patients. Four were heterozygous mutations in four unrelated individuals, belonging to this cohort: c.563G>T in exon 5; c.1169-5T>G in intron 10; c.1251+1G>A in intron 11, and c.1931-2A>G in intron 16. These variants were expressed in HEK 293 cells, and aberrant splicing was verified by in vitro transcription and sequencing of the cDNA. Our findings confirm the need to further investigate the nature of rearrangements associated with this class of mutations and their effects on mature transcripts. In particular, splicing mutations predicted to generate in-frame transcripts may generate out-of-frame mRNA transcripts that do not produce functional ClC-1. Clinically, incomplete molecular evaluation could lead to delayed or faulty diagnosis. PMID:24452722
Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene
Conboy, John G.; Parra, Marilyn K.; Tan, Jeff S.; Mohandas, Narla; Conboy, John G.
In the protein 4.1R gene, alternative first exons splice differentially to alternative 3' splice sites far downstream in exon 2'/2 (E2'/2). We describe a novel intrasplicing mechanism by which exon 1A (E1A) splices exclusively to the distal E2'/2 acceptor via two nested splicing reactions regulated by novel properties of exon 1B (E1B). E1B behaves as an exon in the first step, using its consensus 5' donor to splice to the proximal E2'/2 acceptor. A long region of downstream intron is excised, juxtaposing E1B with E2'/2 to generate a new composite acceptor containing the E1B branchpoint/pyrimidine tract and E2 distal 3' AG-dinucleotide. Next, the upstream E1A splices over E1B to this distal acceptor, excising the remaining intron plus E1B and E2' to form mature E1A/E2 product. We mapped branch points for both intrasplicing reactions and demonstrated that mutation of the E1B 5' splice site or branchpoint abrogates intrasplicing. In the 4.1R gene, intrasplicing ultimately determines N-terminal protein structure and function. More generally, intrasplicing represents a new mechanism whereby alternative promoters can be coordinated with downstream alternative splicing.
Donor site wound protein synthesis correlates with length of acute hospitalization in severely burned children: a pilot study
Tuvdendorj, Demidmaa; Chinkes, David L.; Zhang, Xiao-Jun; Aarsland, Asle; Herndon, David N.
Autografting of burn wounds results in generation of donor site wounds. Here we measured donor site wound protein Fractional Synthesis Rate (FSR) in a burn pediatric population and showed that FSR increases over time postsurgery and correlates with the length of hospital stay (LOS) normalized for total body surface area (TBSA) burn size. 3.9�1.1 days after the grafting surgery patients participated in a metabolic study consisting of continuous infusion of L-[ring-2H5]-phenylalanine and donor site wound punch biopsies. Donor site wound protein FSR was 10.4�7.5 %/day. Wound FSR demonstrated linear correlation with the time postsurgery (p < 0.05). Multiple regression analysis showed that LOS/TBSA correlated with donor site wound protein FSR and time postsurgery (p < 0.001) and the following equation describes the relationship: Estimated LOS/TBSA = (FSR - 12.95 � 1.414 � Postsurgery day)/(?17.8). This equation predicted that FSR corrected for the postsurgery day when the metabolic study was conducted accounted for 67 % of the variability (r2 = 0.673) in the LOS/TBSA. Donor site wound protein FSR correlated to LOS/TBSA of burn patients admitted to the intensive care unit. Measurement of protein deposition in regenerating donor site wound using stable isotope technique provides a quantitative measure of wound healing. PMID:20412556
Allele-specific recognition of the 3? splice site of INS intron 1
Jana Kralovicova; Igor Vorechovsky
Genetic predisposition to type 1 diabetes (T1D) has been associated with a chromosome 11 locus centered on the proinsulin\\u000a gene (INS) and with differential steady-state levels of INS RNA from T1D-predisposing and -protective haplotypes. Here, we show that the haplotype-specific expression is determined\\u000a by INS variants that control the splicing efficiency of intron 1. The adenine allele at IVS1-6 (rs689),
Free Flap Donor Site Reconstruction: A Prospective Case Series Using an Optimized Polyurethane Biodegradable Temporizing Matrix
Wagstaff, Marcus J. D.; Schmitt, Bradley J.; Caplash, Yugesh
Introduction: We recently published a 10-patient case series where free flap donor site reconstruction was performed as a 2-stage procedure using an integrating biodegradable polyurethane matrix (to form a neodermis), followed by definitive closure with an autologous split-skin graft. Two issues were revealed by this pilot study that led to further modification of the biodegradable temporizing matrix. This involved alterations to the seal thickness and bonding to the foam matrix and the introduction of fenestrations to the seal. Objective: This article documents a second cohort of patients requiring free flap (fibular and radial forearm) donor site reconstruction with this optimized material. Methods: The biodegradable temporizing matrix was implanted when the free flap was detached from its donor site. Subsequent integration was monitored closely. Five weeks was the usual time of integration before delamination (seal removal), dermabrasion, and definitive closure with autograft. Results: Integration was complete and uncomplicated in every case, delamination occurred in 1 piece in 1 action, and subsequent graft take was 100% for every patient. Long-term scar outcomes improved compared with the pilot group. Degradation is complete by 12 months, other than occasional microscopic remnants undergoing phagocytosis. Conclusion: This study has reiterated that the biodegradable temporizing matrix can be implanted into humans, followed by neovascularization and integration. No infection was observed, and split-skin overgrafting was successful and uncomplicated.
Solid phase electron donors control denitrification in groundwater at agricultural sites
NASA Astrophysics Data System (ADS)
Green, C. T.; Liao, L.; Bekins, B. A.; Bohlke, J. K.
Increased concentrations of nitrate in groundwater caused by agricultural use of chemical and organic fertilizers are a concern because of possible risks to environmental and human health. At many sites, these problems are mitigated by natural attenuation of nitrate as a result of microbially mediated denitrification of nitrate to nitrogen gas. Recent studies have clarified the factors affecting the rates and extents of denitrification in groundwater in agricultural areas. Intensive studies were conducted by the US Geological Survey to study agricultural chemicals in California, Nebraska, Washington, and Maryland using laboratory analyses, field measurements, and flow and transport modeling for monitoring well transects (0.5 to 2.5 km in length) and vertical profiles (0 to 50 m in depth). Groundwater analyses included major ion chemistry, dissolved gases, nitrogen and oxygen stable isotopes, and atmospheric age-tracers. Sediments were analyzed for concentrations of potential electron donors for denitrification, including reduced iron and sulfur, and organic carbon. Geochemical data and mass balance calculations indicated that solid-phase electron donors were an important factor controlling denitrification at these sites. To examine the generality of this result, a mathematical model of vertical flux of water, oxygen, and nitrate was developed and applied at these study sites along with 2 new study sites in Iowa and Mississippi and 8 additional sites from previous studies in Nebraska, Texas, Minnesota, Wisconsin, North Carolina, Maryland (2 sites), and New York. Model results confirmed the importance of solid phase electron donors. The normalized reaction rates on an electron flux basis tended to increase with depth from the shallow oxygen reduction zone to the underlying nitrate reduction zone. The pattern of higher rates at depth is consistent with a reaction rate controlled by solid phase donors that are depleted under oxidizing conditions near the surface and in greater supply at depth. The eventual depth and rate of migration of nitrate and oxygen in aquifers will depend on the concentrations and reactivities of solid electron donor phases currently in the reduced zones.
Evaluation of an oxygen-diffusion dressing for accelerated healing of donor-site wounds.
Lairet, Kimberly F; Baer, David; Leas, Michelle L; Renz, Evan M; Cancio, Leopoldo C
Accelerating the healing process and reducing pain during healing are beneficial for the following reasons: faster return to work, lower risk of wound infection, improved quality of life, and possibly reduced need for analgesia. This clinical study assessed the effectiveness of a new oxygen-diffusion dressing (OxyBand; Oxyband Technologies, St. Louis, MO) compared with standard Xeroform gauze dressings (Convidien, Mansfield, MA), in the care of skin-graft donor sites in burn patients. Time to healing was the primary endpoint, and pain scores and cosmetic outcome were also assessed. This was a prospective, randomized, controlled study of burn patients undergoing harvesting of two donor sites. Patients were followed at predetermined time points for 30 to 45 days to determine the time to reepithelialization, cosmetic appearance, and pain. Subjects were adult burn patients with less than 30% TBSA burns admitted to the burn center, who required excision and grafting. Twenty patients were enrolled, of whom 17 completed the study. Average age was 35 years. Average burn size was 9.2% TBSA. Patients underwent harvesting of split-thickness skin grafts with one donor wound dressed with OxyBand and the other dressed in Xeroform gauze. Wounds were inspected and photographed on postoperative days 4 and 8, and then every 2 days until the donor wounds were healed. Pain scores at each site were also collected at these visits (rated by patients on a scale from 0 to 10). Mean time to wound healing for OxyBand was 9.3 � 1.7 days; for Xeroform, 12.4 � 2.7 days (P < .001). Pain scores were lower (P < .01) at the OxyBand site compared with the Xeroform site at all time points during postoperative days 4 to 12. There was no difference in the cosmetic outcome of the wounds at 30 to 45 days postoperatively. This study revealed a decrease in the time to healing and in pain at donor sites dressed with an oxygen-diffusion dressing. PMID:23877142
Functional correction by antisense therapy of a splicing mutation in the GALT gene.
Coelho, Ana I; Louren�o, S�lvia; Trabuco, Matilde; Silva, Maria Jo�o; Oliveira, Anabela; Gaspar, Ana; Diogo, Lu�sa; Tavares de Almeida, Isabel; Vicente, Jo�o B; Rivera, Isabel
In recent years, antisense therapy has emerged as an increasingly important therapeutic approach to tackle several genetic disorders, including inborn errors of metabolism. Intronic mutations activating cryptic splice sites are particularly amenable to antisense therapy, as the canonical splice sites remain intact, thus retaining the potential for restoring constitutive splicing. Mutational analysis of Portuguese galactosemic patients revealed the intronic variation c.820+13A>G as the second most prevalent mutation, strongly suggesting its pathogenicity. The aim of this study was to functionally characterize this intronic variation, to elucidate its pathogenic molecular mechanism(s) and, ultimately, to correct it by antisense therapy. Minigene splicing assays in two distinct cell lines and patients' transcript analyses showed that the mutation activates a cryptic donor splice site, inducing an aberrant splicing of the GALT pre-mRNA, which in turn leads to a frameshift with inclusion of a premature stop codon (p.D274Gfs*17). Functional-structural studies of the recombinant wild-type and truncated GALT showed that the latter is devoid of enzymatic activity and prone to aggregation. Finally, two locked nucleic acid oligonucleotides, designed to specifically recognize the mutation, successfully restored the constitutive splicing, thus establishing a proof of concept for the application of antisense therapy as an alternative strategy for the clearly insufficient dietary treatment in classic galactosemia. PMID:25052314
Novel compound heterozygous Thyroglobulin mutations c.745+1G>A/c.7036+2T>A associated with congenital goiter and hypothyroidism in a Vietnamese family. Identification of a new cryptic 5' splice site in the exon 6.
Citterio, Cintia E; Morales, Cecilia M; Bouhours-Nouet, Natacha; Machiavelli, Gloria A; Bueno, Elena; Gatelais, Fr�d�rique; Coutant, Regis; Gonz�lez-Sarmiento, Rogelio; Rivolta, Carina M; Targovnik, H�ctor M
Several patients were identified with dyshormonogenesis caused by mutations in the thyroglobulin (TG) gene. These defects are inherited in an autosomal recessive manner and affected individuals are either homozygous or compound heterozygous for the mutations. The aim of the present study was to identify new TG mutations in a patient of Vietnamese origin affected by congenital hypothyroidism, goiter and low levels of serum TG. DNA sequencing identified the presence of compound heterozygous mutations in the TG gene: the maternal mutation consists of a novel c.745+1G>A (g.IVS6?+?1G>A), whereas the hypothetical paternal mutation consists of a novel c.7036+2T>A (g.IVS40?+?2T>A). The father was not available for segregation analysis. Ex-vivo splicing assays and subsequent RT-PCR analyses were performed on mRNA isolated from the eukaryotic-cells transfected with normal and mutant expression vectors. Minigene analysis of the c.745+1G>A mutant showed that the exon 6 is skipped during pre-mRNA splicing or partially included by use of a cryptic 5' splice site located to 55 nucleotides upstream of the authentic exon 6/intron 6 junction site. The functional analysis of c.7036+2T>A mutation showed a complete skipping of exon 40. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool NNSplice, Fsplice, SPL, SPLM and MaxEntScan programs were investigated and evaluated in relation with the experimental evidence. These analyses predicted that both mutant alleles would result in the abolition of the authentic splice donor sites. The c.745+1G>A mutation originates two putative truncated proteins of 200 and 1142 amino acids, whereas c.7036+2T>A mutation results in a putative truncated protein of 2277 amino acids. In conclusion, we show that the c.745+1G>A mutation promotes the activation of a new cryptic donor splice site in the exon 6 of the TG gene. The functional consequences of these mutations could be structural changes in the protein molecule that alter the biosynthesis of thyroid hormones. PMID:25633667
Evidence for widespread association of mammalian splicing and conserved long-range RNA structures
Pervouchine, Dmitri D.; Khrameeva, Ekaterina E.; Pichugina, Marina Yu.; Nikolaienko, Oleksii V.; Gelfand, Mikhail S.; Rubtsov, Petr M.; Mironov, Andrei A.
Pre-mRNA structure impacts many cellular processes, including splicing in genes associated with disease. The contemporary paradigm of RNA structure prediction is biased toward secondary structures that occur within short ranges of pre-mRNA, although long-range base-pairings are known to be at least as important. Recently, we developed an efficient method for detecting conserved RNA structures on the genome-wide scale, one that does not require multiple sequence alignments and works equally well for the detection of local and long-range base-pairings. Using an enhanced method that detects base-pairings at all possible combinations of splice sites within each gene, we now report RNA structures that could be involved in the regulation of splicing in mammals. Statistically, we demonstrate strong association between the occurrence of conserved RNA structures and alternative splicing, where local RNA structures are generally more frequent at alternative donor splice sites, while long-range structures are more associated with weak alternative acceptor splice sites. As an example, we validated the RNA structure in the human SF1 gene using minigenes in the HEK293 cell line. Point mutations that disrupted the base-pairing of two complementary boxes between exons 9 and 10 of this gene altered the splicing pattern, while the compensatory mutations that reestablished the base-pairing reverted splicing to that of the wild-type. There is statistical evidence for a Dscam-like class of mammalian genes, in which mutually exclusive RNA structures control mutually exclusive alternative splicing. In sum, we propose that long-range base-pairings carry an important, yet unconsidered part of the splicing code, and that, even by modest estimates, there must be thousands of such potentially regulatory structures conserved throughout the evolutionary history of mammals. PMID:22128342
NASA Astrophysics Data System (ADS)
Mudaber, Mohammad Hassan; Yusof, Yuhani
The study of the biological process of deoxyribonucleic acid (DNA) splicing system in a translucent approach was investigated in 2012 by Yusof under the framework of formal language theory. In this work, the concepts of splicing system in two stages as well as splicing languages are mathematically and biologically discussed. Additionally, some theorems based on recognition site factor of initial strings at the existence of two initial strings and two rules are provided via Yusof-Goode (Y-G) approach. Besides, an example is also given in showing the biological meaning of the introduced concept.
Rhodopsin mutations in a Scottish retinitis pigmentosa population, including a novel splice site mutation in intron four.
Bell, C; Converse, C A; Hammer, H M; Osborne, A; Haites, N E
Retinitis pigmentosa (RP) is the name given to a group of disorders, both clinically and genetically heterogeneous, that primarily affect the photoreceptor function of the eye. Mutations in the genes encoding for rhodopsin, RDS-peripherin, or the beta subunit of the cGMP phosphodiesterase enzyme can be responsible for the phenotype. In this study the rhodopsin gene has been screened for mutations in a panel of RP individuals and five different sequence changes have been detected to date in three dominantly inherited and two unclassified families. One of these, a base substitution in the 3'UTR, has not yet been confirmed as disease specific, while three missense substitutions have previously been reported and are likely to be responsible for the phenotype. The fifth change, a base substitution at the intron 4 acceptor splice site, represents a novel mutation and is assumed to be the causative mutation. Images PMID:7819178
Splice acceptor site mutation of the transporter associated with antigen processing-1 gene in human bare lymphocyte syndrome
Furukawa, Hiroshi; Murata, Shigeo; Yabe, Toshio; Shimbara, Naoki; Keicho, Naoto; Kashiwase, Kouichi; Watanabe, Kaoru; Ishikawa, Yoshihide; Akaza, Tatsuya; Tadokoro, Kenji; Tohma, Shigeto; Inoue, Tetsufumi; Tokunaga, Katsushi; Yamamoto, Kazuhiko; Tanaka, Keiji; Juji, Takeo
Expression of histocompatibility leukocyte antigen (HLA) class I molecules on the cell surface depends on the heterodimer of the transporter associated with antigen processing 1 and 2 (TAP1 and TAP2), which transport peptides cleaved by proteasome to the class I molecules. Defects in the TAP2 protein have been reported in two families with HLA class I deficiency, the so-called bare lymphocyte syndrome (BLS) type I. We have, to our knowledge, identified for the first time a splice site mutation in the TAP1 gene of another BLS patient. In addition, class I heavy chains (HCs) did not form the normal complex with tapasin in the endoplasmic reticulum (ER) of the cells of our patient. J. Clin. Invest. 103:649�652 (1999) PMID:10074494
Computational investigation of B-site donor doping effect on fatigue behavior of lead zirconate titanate
NASA Astrophysics Data System (ADS)
Zhang, Zhen; Lu, Li; Shu, Chang; Wu, Ping
The effect of the B-site donor on the fatigue behavior of lead zirconate titanate (PZT) was studied. Seven elements were considered. The formation energies of oxygen vacancies and band structures of these doped systems were investigated. It was found that the Nb, Ta, and W doped PZT systems have the highest formation energy of oxygen vacancies as well as reduced occupations of Ti 3d states by the electrons released from oxygen vacancies. Therefore, the systems respectively doped with Nb, Ta, and W can effectively improve the fatigue property of PZT.
[Functional characterization of two novel splicing mutations in glucoki nase gene in monogenic diabetes MODY2].
Two novel mutations in glucokinase (GCK) gene-G to C substitution at -1 position of intron 7 acceptor splice site (c. 864-1G>C) and synonymous substitution c. 666C>G (GTC>GTG, p.V222V) in exon 6--were identified in patients with monogenic diabetes MODY2 (Maturity Onset Diabetes of Young). GCK minigenes with these mutations were constructed. Analysis of splicing products upon transfection of minigenes into human embryonic cell line HEK293 has shown that each of these nucleotide substitutions impair normal splicing. Mutation c.864-1G>C blocks the usage of normal acceptor site which activates cryptic acceptor splice sites within intron 7 and generates aberrant RNAs containing the portions ofintron 7. Synonymous substitution c.666C>G creates novel donor splice site in exon 6 that leads to formation of defective GCK mRNA with deletion of 16 nucleotides of exon 6. Analysis of in vitro splicing of minigenes confirms the inactivating action of novel mutations on glucokinase expression. PMID:25850297
NOTCH2 and FLT3 gene mis-splicings are common events in patients with acute myeloid leukemia (AML): new potential targets in AML
Bar-Natan, Michal; Haibe-Kains, Benjamin; Pilarski, Patrick M.; Bach, Christian; Pevzner, Samuel; Calimeri, Teresa; Avet-Loiseau, Herve; Lode, Laurence; Verselis, Sigitas; Fox, Edward A.; Galinsky, Ilene; Mathews, Steven; Dagogo-Jack, Ibiayi; Wadleigh, Martha; Steensma, David P.; Motyckova, Gabriela; Deangelo, Daniel J.; Quackenbush, John; Tenen, Daniel G.; Stone, Richard M.; Griffin, James D.
Our previous studies revealed an increase in alternative splicing of multiple RNAs in cells from patients with acute myeloid leukemia (AML) compared with CD34+ bone marrow cells from normal donors. Aberrantly spliced genes included a number of oncogenes, tumor suppressor genes, and genes involved in regulation of apoptosis, cell cycle, and cell differentiation. Among the most commonly mis-spliced genes (>70% of AML patients) were 2, NOTCH2 and FLT3, that encode myeloid cell surface proteins. The splice variants of NOTCH2 and FLT3 resulted from complete or partial exon skipping and utilization of cryptic splice sites. Longitudinal analyses suggested that NOTCH2 and FLT3 aberrant splicing correlated with disease status. Correlation analyses between splice variants of these genes and clinical features of patients showed an association between NOTCH2-Va splice variant and overall survival of patients. Our results suggest that NOTCH2 and FLT3 mis-splicing is a common characteristic of AML and has the potential to generate transcripts encoding proteins with altered function. Thus, splice variants of these genes might provide disease markers and targets for novel therapeutics. PMID:24574459
Alternative splicing and retinal degeneration
Liu, Melissa M.; Zack, Donald J.
Alternative splicing is highly regulated in tissue-specific and development-specific patterns, and it has been estimated that 15% of disease-causing point mutations affect pre-mRNA splicing. In this review, we consider the cis-acting splice site and trans-acting splicing factor mutations that affect pre-mRNA splicing and contribute to retinal degeneration. Numerous splice site mutations have been identified in retinitis pigmentosa and various cone-rod dystrophies. For example, mutations in alternatively spliced retina-specific exons of the widely expressed RPGR and COL2A1 genes lead primarily to X-linked retinitis pigmentosa and ocular variants of Stickler Syndrome, respectively. Furthermore, mutations in general pre-mRNA splicing factors, such as PRPF31, PRPF8, and PRPF3, predominantly cause autosomal dominant retinitis pigmentosa. These findings suggest an important role for pre-mRNA splicing in retinal homeostasis and the pathogenesis of retinal degenerative diseases. The development of novel therapeutic strategies to modulate aberrant splicing, including small molecule based therapies, has the potential to lead to the development of new treatments for retinal degenerative diseases. PMID:23647439
Role of helical constraints of the EBS1�IBS1 duplex of a group II intron on demarcation of the 5? splice site
Popovi?, Milena; Greenbaum, Nancy L.
Recognition of the 5? splice site by group II introns involves pairing between an exon binding sequence (EBS) 1 within the ID3 stem�loop of domain 1 and a complementary sequence at the 3? end of exon 1 (IBS1). To identify the molecular basis for splice site definition of a group IIB ai5? intron, we probed the solution structure of the ID3 stem�loop alone and upon binding of its IBS1 target by solution NMR. The ID3 stem was structured. The base of the ID3 loop was stacked but displayed a highly flexible EBS1 region. The flexibility of EBS1 appears to be a general feature of the ai5? and the smaller Oceanobacillus iheyensis (O.i.) intron and may help in effective search of conformational space and prevent errors in splicing as a result of fortuitous base-pairing. Binding of IBS1 results in formation of a structured seven base pair duplex that terminates at the 5? splice site in spite of the potential for additional A-U and G�U pairs. Comparison of these data with conformational features of EBS1�IBS1 duplexes extracted from published structures suggests that termination of the duplex and definition of the splice site are governed by constraints of the helical geometry within the ID3 loop. This feature and flexibility of the uncomplexed ID3 loop appear to be common for both the ai5? and O.i. introns and may help to fine-tune elements of recognition in group II introns. PMID:24243113
Analysis of a splice-site mutation in the sap-precursor gene of a patient with metachromatic leukodystrophy
Henseler, M.; Klein, A.; Reber, M. [Universitaet Bonn (Germany)] [and others
Sphingolipid activator proteins (SAPs) are small, nonenzymatic glycoproteins required for the lysosomal degradation of various sphingolipids with a short oligosaccharide chain by their exohydrolases. Four of the five known activator proteins (sap-A-sap-D), also called {open_quotes}saposins,{close_quotes} are derived from a common precursor by proteolytic processing. sap-B stimulates hydrolysis of sulfatides by arylsulfatase A in vivo. Its recessively inherited deficiency results in a metabolic disorder similar to classical metachromatic leukodystrophy, which is caused by a defect of arylsulfatase A. Here we report on a patient with sap-B deficiency. Reverse-transcription-PCR studies on the patient`s mRNA revealed the occurrence of two distinct mutant species: one with an in-frame deletion of the first 21 bases of exon 6, the other with a complete in-frame deletion of this exon. The patient was homozygous for the underlying mutation, which was found to be a G-{yields}T transversion within the acceptor splice site between intron e and exon 6, abolishing normal RNA splicing. Allele-specific oligonucleotide hybridization revealed that the parents and both grandfathers of the patient were carriers of this mutation. In order to analyze the fate of the mutant precursor proteins, both abnormal cDNAs were stably expressed in baby hamster kidney cells. Pulse-chase experiments showed that the deletion of 21 bp had no effect on the transport and the maturation of the encoded precursor. All sap forms except sap-B were detectable by immunochemical methods. The cDNA bearing a complete deletion of exon 6 encoded a shortened precursor of only 60 kD, and no mature SAPs were detectable. The carbohydrate chains of this polypeptide were of the high-mannose and hybrid type, indicating no transport of the mutant precursor beyond early Golgi apparatus. An endoplasmic-reticulum localization of this polypeptide was supported by indirect immunofluorescence analysis. 31 refs., 8 figs.
A Spontaneous Fatp4/Scl27a4 Splice Site Mutation in a New Murine Model for Congenital Ichthyosis
Tao, Jianning; Koster, Maranke I.; Harrison, Wilbur; Moran, Jennifer L.; Beier, David R.; Roop, Dennis R.; Overbeek, Paul A.
Congenital ichthyoses are life-threatening conditions in humans. We describe here the identification and molecular characterization of a novel recessive mutation in mice that results in newborn lethality with severe congenital lamellar ichthyosis. Mutant newborns have a taut, shiny, non-expandable epidermis that resembles cornified manifestations of autosomal-recessive congenital ichthyosis in humans. The skin is stretched so tightly that the newborn mice are immobilized. The genetic defect was mapped to a region near the proximal end of chromosome 2 by SNP analysis, suggesting Fatp4/Slc27a4 as a candidate gene. FATP4 mutations in humans cause ichthyosis prematurity syndrome (IPS), and mutations of Fatp4 in mice have previously been found to cause a phenotype that resembles human congenital ichthyoses. Characterization of the Fatp4 cDNA revealed a fusion of exon 8 to exon 10, with deletion of exon 9. Genomic sequencing identified an A to T mutation in the splice donor sequence at the 3?-end of exon 9. Loss of exon 9 results in a frame shift mutation upstream from the conserved very long-chain acyl-CoA synthase (VLACS) domain. Histological studies revealed that the mutant mice have defects in keratinocyte differentiation, along with hyperproliferation of the stratum basale of the epidermis, a hyperkeratotic stratum corneum, and reduced numbers of secondary hair follicles. Since Fatp4 protein is present primarily at the stratum granulosum and the stratum spinosum, the hyperproliferation and the alterations in hair follicle induction suggest that very long chain fatty acids, in addition to being required for normal cornification, may influence signals from the stratum corneum to the basal cells that help to orchestrate normal skin differentiation. PMID:23226340
HS3D, A Dataset of Homo Sapiens Splice Regions, and its Extraction Procedure from a Major Public Database
NASA Astrophysics Data System (ADS)
Pollastro, Pasquale; Rampone, Salvatore
The aim of this work is to describe a cleaning procedure of GenBank data, producing material to train and to assess the prediction accuracy of computational approaches for gene characterization. A procedure (GenBank2HS3D) has been defined, producing a dataset (HS3D - Homo Sapiens Splice Sites Dataset) of Homo Sapiens Splice regions extracted from GenBank (Rel.123 at this time). It selects, from the complete GenBank Primate Division, entries of Human Nuclear DNA according with several assessed criteria; then it extracts exons and introns from these entries (actually 4523 + 3802). Donor and acceptor sites are then extracted as windows of 140 nucleotides around each splice site (3799 + 3799). After discarding windows not including canonical GT-AG junctions (65 + 74), including insufficient data (not enough material for a 140 nucleotide window) (686 + 589), including not AGCT bases (29 + 30), and redundant (218 + 226), the remaining windows (2796 + 2880) are reported in the dataset. Finally, windows of false splice sites are selected by searching canonical GT-AG pairs in not splicing positions (271 937 + 332 296). The false sites in a range +/- 60 from a true splice site are marked as proximal. HS3D, release 1.2 at this time, is available at the Web server of the University of Sannio: http://www.sci.unisannio.it/docenti/rampone/.
Changes in type II procollagen isoform expression during chondrogenesis by disruption of an alternative 5� splice site within Col2a1 exon 2
Hering, Thomas M.; Wirthlin, Louisa; Ravindran, Soumya; McAlinden, Audrey
This study describes a new mechanism controlling the production of alternatively-spliced isoforms of type II procollagen (Col2a1) in vivo. During chondrogenesis, precursor chondrocytes predominantly produce isoforms containing alternatively-spliced exon 2 (type IIA and IID) while Col2a1 mRNA devoid of exon 2 (type IIB) is the major isoform produced by differentiated chondrocytes. We previously identified an additional Col2a1 isoform containing a truncated exon 2 and premature termination codons in exon 6 (type IIC). This transcript is produced by utilization of another 5� splice site present in exon 2. To determine the role of this IIC splicing event in vivo, we generated transgenic mice containing silent knock-in mutations at the IIC 5� splice site (Col2a1-mIIC), thereby inhibiting production of IIC transcripts. Heterozygous and homozygous knock-in mice were viable and display no overt skeletal phenotype to date. However, RNA expression profiles revealed that chondrocytes in cartilage from an age range of Col2a1-mIIC mice produced higher levels of IIA and IID mRNAs and decreased levels of IIB mRNAs throughout pre-natal and post-natal development, when compared to chondrocytes from littermate control mice. Immunofluorescence analyses showed a clear increase in expression of embryonic type II collagen protein isoforms (i.e. containing the exon 2-encoded cysteine-rich (CR) protein domain) in cartilage extracellular matrix (ECM). Interestingly, at P14, P28 and P56, expression of embryonic Col2a1 isoforms in Col2a1-mIIC mice persisted in the pericellular domain of the ECM in articular and growth plate cartilage. We also show that persistent expression of the exon 2-encoded CR domain in the ECM of post-natal cartilage tissue may be due, in part, to the embryonic form of type XI collagen (the ?3 chain of which is also encoded by the Col2a1 gene). In conclusion, expression of the Col2a1 IIC splice form may have a regulatory function in controlling alternative splicing of exon 2 to generate defined proportions of IIA, IID and IIB procollagen isoforms during cartilage development. Future studies will involve ultrastructural and biomechanical analysis of the collagen ECM to determine the effects of persistent mis-expression of embryonic collagen isoforms in mature cartilage tissue. PMID:24735995
Xanthogranulomatous inflammation involving latissimus dorsi donor site and implant breast reconstruction: case report and literature review.
Hussain, Tasadooq; Elahi, Bilal; Long, Ervine; Mahapatra, Tapan; McManus, Penelope L; Kneeshaw, Peter J
Xanthogranulomatous inflammation is a rare clinico-pathological condition involving many organ systems. Breast involvement with this rare condition reported from a few cases of mastitis has been limited to only microscopic involvement on histology. We would like to report an unusual presentation of this inflammatory process presenting as a solid lump mimicking malignancy in latissimus dorsi donor site scar and implant-based breast reconstruction as a result of a ruptured silicone gel implant. To our knowledge there have been no previous reports on similar presentation published in the literature. This case highlights a rare complication of a leaked silicone gel implant triggering a xanthomatous response in the absence of the usual infective or obstructing etiologies. This condition is of benign nature with complete clearance on surgical excision and excellent clinical prognosis reported from other organ involvement. PMID:22906098
Xanthogranulomatous inflammation involving latissimus dorsi donor site and implant breast reconstruction: case report and literature review
Xanthogranulomatous inflammation is a rare clinico-pathological condition involving many organ systems. Breast involvement with this rare condition reported from a few cases of mastitis has been limited to only microscopic involvement on histology. We would like to report an unusual presentation of this inflammatory process presenting as a solid lump mimicking malignancy in latissimus dorsi donor site scar and implant-based breast reconstruction as a result of a ruptured silicone gel implant. To our knowledge there have been no previous reports on similar presentation published in the literature. This case highlights a rare complication of a leaked silicone gel implant triggering a xanthomatous response in the absence of the usual infective or obstructing etiologies. This condition is of benign nature with complete clearance on surgical excision and excellent clinical prognosis reported from other organ involvement. PMID:22906098
In silico and in vivo splicing analysis of MLH1 and MSH2 missense mutations shows exon- and tissue-specific effects
Lastella, Patrizia; Surdo, Nicoletta Concetta; Resta, Nicoletta; Guanti, Ginevra; Stella, Alessandro
Background Abnormalities of pre-mRNA splicing are increasingly recognized as an important mechanism through which gene mutations cause disease. However, apart from the mutations in the donor and acceptor sites, the effects on splicing of other sequence variations are difficult to predict. Loosely defined exonic and intronic sequences have been shown to affect splicing efficiency by means of silencing and enhancement mechanisms. Thus, nucleotide substitutions in these sequences can induce aberrant splicing. Web-based resources have recently been developed to facilitate the identification of nucleotide changes that could alter splicing. However, computer predictions do not always correlate with in vivo splicing defects. The issue of unclassified variants in cancer predisposing genes is very important both for the correct ascertainment of cancer risk and for the understanding of the basic mechanisms of cancer gene function and regulation. Therefore we aimed to verify how predictions that can be drawn from in silico analysis correlate with results obtained in an in vivo splicing assay. Results We analysed 99 hMLH1 and hMSH2 missense mutations with six different algorithms. Transfection of three different cell lines with 20 missense mutations, showed that a minority of them lead to defective splicing. Moreover, we observed that some exons and some mutations show cell-specific differences in the frequency of exon inclusion. Conclusion Our results suggest that the available algorithms, while potentially helpful in identifying splicing modulators especially when they are located in weakly defined exons, do not always correspond to an obvious modification of the splicing pattern. Thus caution must be used in assessing the pathogenicity of a missense or silent mutation with prediction programs. The variations observed in the splicing proficiency in three different cell lines suggest that nucleotide changes may dictate alternative splice site selection in a tissue-specific manner contributing to the widely observed phenotypic variability in inherited cancers. PMID:16995940
A Biodegradable Polyurethane Dermal Matrix in Reconstruction of Free Flap Donor Sites: A Pilot Study
Wagstaff, Marcus J.D.; Schmitt, Bradley J.; Coghlan, Patrick; Finkemeyer, James P.; Caplash, Yugesh
We have developed a biodegradable temporizing matrix (BTM) capable of supporting secondary split-skin graft-take in animal studies. We report its first long-term implantation and use as a dermal scaffold in humans. This preliminary study assesses its ability to integrate, its ease of delamination, its ability to sustain split-skin graft in complex wounds, the degree of wound contraction, and ultimately the quality of the scar at 1 year postimplantation. Ten patients were recruited, each requiring elective free flap reconstruction. Free flap donor sites created were anterolateral thigh flaps, fibular osseocutaneous flaps, or radial/ulnar forearm (RF/UF) flaps. The BTM was implanted when the flap was detached from its donor site. Dressing changes were performed twice weekly. The time elapsed between implantation and delamination depended on the type of flap and thus the wound bed left. Once integrated, the BTMs were delaminated in theatre, and the surface of the �neodermis� was refreshed by dermabrasion, prior to application of a split-skin graft. The BTM integration occurred in all patients (100% in 6 patients, with 90%, 84%, 76%, and 60% integration in the remainder). Integrated BTM sustained successful graft-take in all patients. Complete take was marred in 2 patients, over areas of BTM that had not integrated and graft application was performed too early. The BTM can be applied into wounds in humans and can integrate, persist in the presence of infection, and sustain split-skin overgrafting, despite the trial group presenting with significant comorbidities. PMID:25987938
A biodegradable polyurethane dermal matrix in reconstruction of free flap donor sites: a pilot study.
Wagstaff, Marcus J D; Schmitt, Bradley J; Coghlan, Patrick; Finkemeyer, James P; Caplash, Yugesh; Greenwood, John E
We have developed a biodegradable temporizing matrix (BTM) capable of supporting secondary split-skin graft-take in animal studies. We report its first long-term implantation and use as a dermal scaffold in humans. This preliminary study assesses its ability to integrate, its ease of delamination, its ability to sustain split-skin graft in complex wounds, the degree of wound contraction, and ultimately the quality of the scar at 1 year postimplantation. Ten patients were recruited, each requiring elective free flap reconstruction. Free flap donor sites created were anterolateral thigh flaps, fibular osseocutaneous flaps, or radial/ulnar forearm (RF/UF) flaps. The BTM was implanted when the flap was detached from its donor site. Dressing changes were performed twice weekly. The time elapsed between implantation and delamination depended on the type of flap and thus the wound bed left. Once integrated, the BTMs were delaminated in theatre, and the surface of the "neodermis" was refreshed by dermabrasion, prior to application of a split-skin graft. The BTM integration occurred in all patients (100% in 6 patients, with 90%, 84%, 76%, and 60% integration in the remainder). Integrated BTM sustained successful graft-take in all patients. Complete take was marred in 2 patients, over areas of BTM that had not integrated and graft application was performed too early. The BTM can be applied into wounds in humans and can integrate, persist in the presence of infection, and sustain split-skin overgrafting, despite the trial group presenting with significant comorbidities. PMID:25987938
Mutations in the Caenorhabditis elegans U2AF Large Subunit UAF-1 Al= of a 3' Splice Site In Vivo
Ma, Long
The removal of introns from eukaryotic RNA transcripts requires the activities of five multi-component ribonucleoprotein complexes and numerous associated proteins. The lack of mutations affecting splicing factors essential ...
The spfash mouse: a missense mutation in the ornithine transcarbamylase gene also causes aberrant mRNA splicing.
Hodges, P E; Rosenberg, L E
Ornithine transcarbamylase (ornithine carbamoyltransferase; carbamoyl-phosphate:L-ornithine carbamoyltransferase, EC 126.96.36.199) is a mitochondrial matrix enzyme of the mammalian urea cycle. The X chromosome-linked spfash mutation in the mouse causes partial ornithine transcarbamylase deficiency and has served as a model for the human disease. We show here that the spfash mutation is a guanine to adenine transition of the last nucleotide of the fourth exon of the ornithine transcarbamylase gene. This nucleotide change produces two remarkably different effects. First, this transition causes ornithine transcarbamylase mRNA deficiency because the involved exon nucleotide plays a part in the recognition of the adjacent splice donor site. As a result of the mutation, ornithine transcarbamylase pre-mRNA is spliced inefficiently both at this site and at a cryptic splice donor site 48 bases into the adjacent intron. Second, two mutant proteins are translated from these mRNAs. From the correctly spliced mRNA, the transition results in a change of amino acid 129 from arginine to histidine. This missense substitution has no discernable effect on mitochondrial import, subunit assembly, or enzyme activity. On the other hand, the elongated mRNA resulting from mis-splicing is translated into a dysfunctional ornithine transcarbamylase subunit elongated by the insertion of 16 amino acid residues. Images PMID:2471197
A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection
NASA Astrophysics Data System (ADS)
Elliott, David J.; Bourgeois, Cyril F.; Klink, Albrecht; St�venin, James; Cooke, Howard J.
RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.
Species-Dependent Splice Recognition of a Cryptic Exon Resulting from a Recurrent Intronic CEP290 Mutation that Causes Congenital Blindness
Garanto, Alejandro; Duijkers, Lonneke; Collin, Rob W. J.
A mutation in intron 26 of CEP290 (c.2991+1655A>G) is the most common genetic cause of Leber congenital amaurosis (LCA), a severe type of inherited retinal degeneration. This mutation creates a cryptic splice donor site, resulting in the insertion of an aberrant exon (exon X) into ~50% of all CEP290 transcripts. A humanized mouse model with this mutation did not recapitulate the aberrant CEP290 splicing observed in LCA patients, suggesting differential recognition of cryptic splice sites between species. To further assess this phenomenon, we generated two CEP290 minigene constructs, with and without the intronic mutation, and transfected these in cell lines of various species. RT-PCR analysis revealed that exon X is well recognized by the splicing machinery in human and non-human primate cell lines. Intriguingly, this recognition decreases in cell lines derived from species such as dog and rodents, and it is completely absent in Drosophila. In addition, other cryptic splicing events corresponding to sequences in intron 26 of CEP290 were observed to varying degrees in the different cell lines. Together, these results highlight the complexity of splice site recognition among different species, and show that care is warranted when generating animal models to mimic splice site mutations in vivo. PMID:25761237
A late-onset and mild form of Charcot-Marie-Tooth disease type 2 caused by a novel splice-site mutation within the Mitofusin-2 gene.
Kotruchow, Katarzyna; Kabzi?ska, Dagmara; Hausmanowa-Petrusewicz, Irena; Kocha?ski, Andrzej
Charcot-Marie-Tooth type 2A disease (CMT2A) caused by mutations in the Mitofusin 2 gene (Mfn2) has been shown to be an early-onset axonal neuropathy with severe clinical course in the majority of the patients. In this study we present a unique phenotype of CMT2A disease characterized by late-onset polyneuropathy with a very mild clinical course. This rare form of CMT2A disease is caused by a new splice-site (c.311+1G>T) mutation within the MFN2 gene. Due to disturbance of the MFN2 splicing process, this mutation generates a short transcript which encodes a very short fragment of MFN2 protein. The c.311+1G>T mutation within the MFN2 gene results in the late -onset CMT2 disease. PMID:24803844
The Maturation of Synthetic Scaffolds for Osteochondral Donor Sites of the Knee
Bedi, Asheesh; Foo, Li Foong; Williams, Riley J.; Potter, Hollis G.
Objective: The purpose of this study was to analyze the morphological imaging characteristics and incorporation of TruFit bone graft substitute (BGS) plugs using cartilage-sensitive magnetic resonance imaging (MRI) and quantitative T2 mapping. Design: Twenty-six patients (mean age, 28.72 years; range, 11-56 years) underwent osteochondral autologous transplantation (OATS) for chondral defects with filling of the knee joint donor sites using Trufit BGS plugs. The mean follow-up interval between implantation and MRI analysis was 21.3 months (range, 6-39 months). During this period, 43 cartilage-sensitive and 25 quantitative T2-mapping MRI studies were performed. The donor sites were assessed for plug and interface morphology, displacement, hypertrophy, subchondral edema, presence of bony overgrowth, percentage fill, and degree of incorporation. T2 relaxation times were measured for the superficial and deep layers of the repair tissue. A linear regression and correlational analysis was performed with Bonferroni correction, and P < 0.05 was defined as significant. Results: Longitudinal analysis revealed favorable plug appearance at early follow-up (?6 months), with 75% of plugs demonstrating flush morphology and 78% demonstrating near complete to complete fill. Plug appearance deteriorated at intermediate follow-up (~12 months), with only 26% of plugs demonstrating flush morphology and 52% with near complete or complete fill. Plug appearance substantially improved with longer follow-up (?16 months), with 70% of plugs demonstrating flush morphology and 90% demonstrating near complete or complete fill. Interface resorption was common at ~12 months (P < 0.0001) and was associated with older age (P = 0.01) or a single-plug configuration (P = 0.04). T2 values for the repair cartilage approached that of normal cartilage with increasing duration after surgery (P < 0.004), more so for single- compared with multiple-plug configurations (P = 0.03). Conclusions: The Trufit BGS plug demonstrates a predictable pattern of postoperative maturation on MRI images that parallels its biological incorporation. An intermediate postoperative interval can be associated with unfavorable MRI findings. However, the plug appearance significantly improves with greater postoperative duration and has mean T2 relaxation times that approach those of normal articular cartilage.
Pinpointing retrovirus entry sites in cells expressing alternatively spliced receptor isoforms by single virus imaging
Background The majority of viruses enter host cells via endocytosis. Current knowledge of viral entry pathways is largely based upon infectivity measurements following genetic and/or pharmacological interventions that disrupt vesicular trafficking and maturation. Imaging of single virus entry in living cells provides a powerful means to delineate viral trafficking pathways and entry sites under physiological conditions. Results Here, we visualized single avian retrovirus co-trafficking with markers for early (Rab5) and late (Rab7) endosomes, acidification of endosomal lumen and the resulting viral fusion measured by the viral content release into the cytoplasm. Virus-carrying vesicles either merged with the existing Rab5-positive early endosomes or slowly accumulated Rab5. The Rab5 recruitment to virus-carrying endosomes correlated with acidification of their lumen. Viral fusion occurred either in early (Rab5-positive) or intermediate (Rab5- and Rab7-positive) compartments. Interestingly, different isoforms of the cognate receptor directed virus entry from distinct endosomes. In cells expressing the transmembrane receptor, viruses preferentially entered and fused with slowly maturing early endosomes prior to accumulation of Rab7. By comparison, in cells expressing the GPI-anchored receptor, viruses entered both slowly and quickly maturing endosomes and fused with early (Rab5-positive) and intermediate (Rab5- and Rab7-positive) compartments. Conclusions Since the rate of low pH-triggered fusion was independent of the receptor isoform, we concluded that the sites of virus entry are determined by the kinetic competition between endosome maturation and viral fusion. Our findings demonstrate the ability of this retrovirus to enter cells via alternative endocytic pathways and establish infection by releasing its content from distinct endosomal compartments. PMID:24935247
A sequence compilation and comparison of exons that are alternatively spliced in neurons.
Stamm, S; Zhang, M Q; Marr, T G; Helfman, D M
Alternative splicing is an important regulatory mechanism to create protein diversity. In order to elucidate possible regulatory elements common to neuron specific exons, we created and statistically analysed a database of exons that are alternatively spliced in neurons. The splice site comparison of alternatively and constitutively spliced exons reveals that some, but not all alternatively spliced exons have splice sites deviating from the consensus sequence, implying diverse patterns of regulation. The deviation from the consensus is most evident at the -3 position of the 3' splice site and the +4 and -3 position of the 5' splice site. The nucleotide composition of alternatively and constitutively spliced exons is different, with alternatively spliced exons being more AU rich. We performed overlapping k-tuple analysis to identify common motifs. We found that alternatively and constitutively spliced exons differ in the frequency of several trinucleotides that cannot be explained by the amino acid composition and may be important for splicing regulation. PMID:8202349
NMR structure of the 5? splice site in the group IIB intron Sc.ai5?�conformational requirements for exon�intron recognition
Kruschel, Daniela; Skilandat, Miriam; Sigel, Roland K.O.
A crucial step of the self-splicing reaction of group II intron ribozymes is the recognition of the 5? exon by the intron. This recognition is achieved by two regions in domain 1 of the intron, the exon-binding sites EBS1 and EBS2 forming base pairs with the intron-binding sites IBS1 and IBS2 located at the end of the 5? exon. The complementarity of the EBS1�IBS1 contact is most important for ensuring site-specific cleavage of the phosphodiester bond between the 5? exon and the intron. Here, we present the NMR solution structures of the d3? hairpin including EBS1 free in solution and bound to the IBS1 7-mer. In the unbound state, EBS1 is part of a flexible 11-nucleotide (nt) loop. Binding of IBS1 restructures and freezes the entire loop region. Mg2+ ions are bound near the termini of the EBS1�IBS1 helix, stabilizing the interaction. Formation of the 7-bp EBS1�IBS1 helix within a loop of only 11 nt forces the loop backbone to form a sharp turn opposite of the splice site, thereby presenting the scissile phosphate in a position that is structurally unique. PMID:24448450
Genotype-phenotype associations in neurofibromatosis type 1 (NF1): an increased risk of tumor complications in patients with NF1 splice-site mutations?
Neurofibromatosis type 1 (NF1) is a complex neurocutaneous disorder with an increased susceptibility to develop both benign and malignant tumors but with a wide spectrum of inter and intrafamilial clinical variability. The establishment of genotype-phenotype associations in NF1 is potentially useful for targeted therapeutic intervention but has generally been unsuccessful, apart from small subsets of molecularly defined patients. The objective of this study was to evaluate the clinical phenotype associated with the specific types of NF1 mutation in a retrospectively recorded clinical dataset comprising 149 NF1 mutation-known individuals from unrelated families. Each patient was assessed for ten NF1-related clinical features, including the number of caf�-au-lait spots, cutaneous and subcutaneous neurofibromas and the presence/absence of intertriginous skin freckling, Lisch nodules, plexiform and spinal neurofibromas, optic gliomas, other neoplasms (in particular CNS gliomas, malignant peripheral nerve sheath tumors (MPNSTs), juvenile myelomonocytic leukemia, rhabdomyosarcoma, phaechromocytoma, gastrointestinal stromal tumors, juvenile xanthogranuloma, and lipoma) and evidence of learning difficulties. Gender and age at examination were also recorded. Patients were subcategorized according to their associated NF1 germ line mutations: frame shift deletions (52), splice-site mutations (23), nonsense mutations (36), missense mutations (32) and other types of mutation (6). A significant association was apparent between possession of a splice-site mutation and the presence of brain gliomas and MPNSTs (p?=?0.006). If confirmed, these findings are likely to be clinically important since up to a third of NF1 patients harbor splice-site mutations. A significant influence of gender was also observed on the number of subcutaneous neurofibromas (females, p?=?0.009) and preschool learning difficulties (females, p?=?0.022). PMID:23244495
Evolution of shorter and more hydrophilic transthyretin N-termini by stepwise conversion of exon 2 into intron 1 sequences (shifting the 3' splice site of intron 1)
Aldred, A R; Prapunpoj, P; Schreiber, G
Transthyretin cDNA was cloned from Eastern Grey Kangaroo liver and its nucleotide sequence determined. Analysis of the derived amino acid sequence of kangaroo transthyretin, together with data obtained previously for transthyretins from other vertebrate species [Duan, W., Richardson, S. J., Babon, J. J., Heyes, R. J., Southwell, B. R., Harms, P. J., Wettenhall, R. E. H., Dziegielewska, K. M., Selwood, L., Bradley, A. J., Brack, C. M. & Schreiber, G. (1995) Eur. J. Biochem. 227, 396-406], showed that the N-terminus is the region which changes most distinctly during evolution. It has been shown for human, mouse and rat transthyretins, that this region is encoded by DNA at the border of exon 1 and exon 2. Therefore, this section of transthyretin genomic DNA was amplified by PCR and directly sequenced for the Buffalo Rat, Tammar Wallaby, Eastern Grey Kangaroo, Stripe-faced Dunnart, Short-tailed Grey Opossum and White Leghorn Chicken. The splice sites at both ends of intron 1 were identified by comparison with the cDNA sequences. The obtained data suggest that the N-termini of transthyretin evolved by successive shifts of the 3' splice site of intron 1 in the 3' direction, resulting in successive shortening of the 5' end of exon 2. At the protein level, this resulted in a shorter and more hydrophilic N-terminal region of transthyretin. Successive shifts in splice sites may be an evolutionary mechanism of general importance, since they can lead to stepwise changes in the properties of proteins. This could be a molecular mechanism for positive Darwinian selection. PMID:9208931
Novel splice site mutations and a large deletion in three patients with the cblF inborn error of vitamin B12 metabolism.
Miousse, Isabelle R; Watkins, David; Rosenblatt, David S
The cblF disorder, characterized by accumulation of internalized cobalamin in the lysosome, is caused by mutations in the LMBRD1 gene which encodes an integral lysosomal membrane protein. We describe novel mutations in LMBRD1 in three patients: two splice site mutations, c.916-1G>T and c.1339-1G>T, and a 6785 bp deletion encompassing exon 2, c.70-4298_246+2311del6785. The three patients are compound heterozygotes for one novel mutation and the common c.1056delG mutation. PMID:21303734
Novel splice site mutations and a large deletion in three patients with the cblF inborn error of vitamin B 12 metabolism
Isabelle R. Miousse; David Watkins; David S. Rosenblatt
The cblF disorder, characterized by accumulation of internalized cobalamin in the lysosome, is caused by mutations in the LMBRD1 gene which encodes an integral lysosomal membrane protein. We describe novel mutations in LMBRD1 in three patients: two splice site mutations, c.916-1G>T and c.1339-1G>T, and a 6785bp deletion encompassing exon 2, c.70-4298_246+2311del6785. The three patients are compound heterozygotes for one novel
Evaluation of Six Split-thickness Skin Graft Donor-site Dressing Materials in a Swine Model
Masella, Pamela C.; Balent, Eric M.; Carlson, Terri L.; Lee, Karen W.
Background: Numerous dressings for split-thickness skin graft donor sites are commercially available with no conclusive evidence-based consensus regarding the optimal dressing choice. This study was conducted to identify which of 5 commonly used materials promotes wound healing most effectively for use on split-thickness donor sites in comparison with our standard dressing, Xeroform (petrolatum gauze). Methods: Twenty-four partial-thickness wounds were created on the backs of 4 pigs using a dermatome. Wounds (n = 4 per dressing type per pig) were treated with Xeroform, Opsite (polyurethane film), Kaltostat ( calcium sodium alginate), DuoDERM (hydrocolloid), Aquacel (hydrofiber), and Mepilex (silicone foam). Full-thickness skin samples were excised at 3 or 5 days and evaluated histologically for reepithelialization and inflammation. Comparisons also included incidence of infection, ease of use, and cost analyses. Results: DuoDERM elicited the greatest percent reepithelialization (81%) and Mepilex the lowest (33%) after 3 days (P = 0.004). All dressings demonstrated complete reepithelialization except Mepilex (85%) at 5 days. There were no infections and inflammation was mild among all treatments. Mepilex was easiest to use, whereas Aquacel, Kaltostat, and Opsite were most difficult (P = 0.03). Xeroform was most cost-effective and Aquacel most expensive. Combined scoring revealed DuoDERM = Xeroform > Opsite = Mepilex > Kaltostat > Aquacel. Conclusions: DuoDERM and Xeroform were most effective overall. DuoDERM tended to outperform all dressings in reepithelialization at 3 days, while Xeroform was least expensive, easy to use, and demonstrated rapid reepithelialization. These findings suggest that Xeroform may be preferred for use on large donor-site areas. DuoDERM may be more appropriate for small donor sites when healing time is a priority. PMID:25289278
Long-term functional donor site morbidity of the free radial forearm flap in head and neck cancer survivors
Background To assess the functional donor site morbidity of the forearm free flap in patients surviving at least 2�years after ablative head and neck cancer surgery in a tertiary care centre. Methods This study involved nine long-term survivors (2�year post-operative) who had forearm free flaps to reconstruct head and neck defects. All flaps were raised from the non-dominant arm. The non-donor side acted as a control for all patients. Objective measurements were as follows: grip, tip pinch and key pinch strength measured with dynamometers; flexion, extension, radial and ulnar deviation and pronation and supination range of motion at the wrist measured with goniometry; A timed manual dexterity task was performed with a grooved pegboard test, and sensation of the radial nerve was tested with Semmes Weinstein monofilaments. Subjective measurements included a validated patient questionnaire of hand function and opinions of scar appearance as well as a validated scar assessment from two different observers. Results Pronation at the wrist, manual dexterity and sensation were found to be significantly reduced in the donor side compared to the non-donor side. Inter-rater agreement between the two observers was found to be poor, except for an acceptable correlation between overall scar opinions. No correlations were found between any subjective or objective items or between the patient�s and the observers� subjective evaluations. Conclusions Donor site morbidity can be demonstrated with objective testing however this is accepted and well tolerated by head and neck cancer patients. PMID:24418459
Evaluation of human amniotic membrane as a wound dressing for split-thickness skin-graft donor sites.
Loeffelbein, Denys J; Rohleder, Nils H; Eddicks, Matthias; Baumann, Claudia M; Stoeckelhuber, Mechthild; Wolff, Klaus-D; Drecoll, Enken; Steinstraesser, Lars; Hennerbichler, Simone; Kesting, Marco R
Human amniotic membrane (HAM) has been used as a biomaterial in various surgical procedures and exceeds some qualities of common materials. We evaluated HAM as wound dressing for split-thickness skin-graft (STSG) donor sites in a swine model (Part A) and a clinical trial (Part B). Part A: STSG donor sites in 4 piglets were treated with HAM or a clinically used conventional polyurethane (PU) foil (n = 8 each). Biopsies were taken on days 5, 7, 10, 20, 40, and 60 and investigated immunohistochemically for alpha-smooth muscle actin (?SMA: wound contraction marker), von Willebrand factor (vWF: angiogenesis), Ki-67 (cell proliferation), and laminin (basement membrane integrity). Part B: STSG donor sites in 45 adult patients (16 female/29 male) were treated with HAM covered by PU foam, solely by PU foam, or PU foil/paraffin gauze (n = 15 each). Part A revealed no difference in the rate of wound closure between groups. HAM showed improved esthetic results and inhibitory effects on cicatrization. Angioneogenesis was reduced, and basement membrane formation was accelerated in HAM group. Part B: no difference in re-epithelialization/infection rate was found. HAM caused less ichor exudation and less pruritus. HAM has no relevant advantage over conventional dressings but might be a cost-effective alternative. PMID:25003117
A randomised controlled pilot study comparing Mepitel(�) and SurfaSoft(�) on paediatric donor sites treated with Recell(�).
Campanella, S D; Rapley, P; Ramelet, A-S
This randomized controlled pilot study examined the effects of a silicone net dressing (Mepitel(�)) and a monofilament polyamide woven dressing (SurfaSoft(�)) on the rate of epithelialisation and epidermal maturation, pain, and ease of dressing removal on paediatric donor sites treated with epithelial cell suspension (ReCell(�)). Fifteen children (1-15 years) admitted for acute or reconstructive burns procedures in a tertiary referral hospital in Australia were randomly assigned to the experimental group, Mepitel(�) (n=8) and to the control group, SurfaSoft(�) (n=7). All donor sites were treated with ReCell(�) and covered with the assigned dressing. Measurements of rate of epithelialisation and epidermal maturation, pain, and ease of dressing removal were recorded every two days until the wound was healed. Results showed that there was no difference in the rate of epidermal maturation between the two groups. Less pain and force to remove the dressing was shown in the Mepitel(�) group when compared to SurfaSoft(�). The rate of epithelialisation was found to be an unreliable measure. Although additional research is required to support the results of this study, these results suggest that Mepitel's(�) pliable, self-adhesive and atraumatic properties may improve healing of ReCell(�) treated donor sites with less pain at dressing changes. This pilot study provides a strong base for further research in this area. PMID:21982622
Multiscale Modeling of Alternative Splicing Regulation
Damien Eveillard; Delphine Ropers; Hidde De Jong; Christiane Branlant; Alexander Bockmayr
Alternative splicing is a key process in post-transcriptional regulation, by which several kinds of mature RNA can be obtained from the same premessenger RNA. Using a constraint programming approach, we model the alternative splicing regulation at different scales (single site vs. multiple sites), thus exploiting different types of available experimen- tal data.
Splice-Site Mutations Cause Rrp6-Mediated Nuclear Retention of the Unspliced RNAs and Transcriptional Down-Regulation of the Splicing-Defective Genes
Andrea B. Eberle; Viktoria Hessle; Roger Helbig; Widad Dantoft; Niclas Gimber; Neus Visa; Yue Feng
BackgroundEukaryotic cells have developed surveillance mechanisms to prevent the expression of aberrant transcripts. An early surveillance checkpoint acts at the transcription site and prevents the release of mRNAs that carry processing defects. The exosome subunit Rrp6 is required for this checkpoint in Saccharomyces cerevisiae, but it is not known whether Rrp6 also plays a role in mRNA surveillance in higher
Objective and subjective evaluation of donor-site morbidity after nipple sharing for nipple areola reconstruction.
Haslik, W; Nedomansky, J; Hacker, S; Nickl, S; Schroegendorfer, K F
Nipple reconstruction is of importance in achieving the best possible aesthetic outcome after breast reconstruction. Nipple sharing is a common technique; this study focused on the potential morbidity at the donor nipple. Between 2008 and 2012, 26 patients underwent nipple sharing at our institution. The donor nipple was examined before and after the procedure (mean follow-up of 21 months). Sensitivity, projection, diameter, and patient satisfaction were evaluated. The sensitivity in the donor nipple decreased, albeit insignificantly, from 1.2 g/mm2 (0.8-1.6) to 1.8 g/mm2 (0.8-4.8) (p=0.054, n=26). The projection due to graft removal decreased from 8.0 mm (6.8-10.0) to 4.5 mm (4.0-5.0) (p=0.001). Of the patients, 88% were "very satisfied" or "somewhat satisfied" with the sensitivity and 89% with the symmetry between the donor and reconstructed nipple. At least 60% of the patients were "very satisfied" with all aesthetic outcome parameters (projection, appearance, naturalness, color, and shape). All patients would agree to undergo this procedure again, if necessary. Nipple sharing was associated with minimal morbidity at the donor nipple. The postoperative projection was adequate. Regardless of whether simultaneous mastopexy was performed, the loss of sensitivity was minimal and presumably imperceptible to the patient. By using no sutures after graft removal and letting the donor nipple heal spontaneously, scarring was minimized and the natural appearance and good sensitivity of the donor nipple were preserved. PMID:25465146
Identification of a novel splice site mutation of CLCN5 gene and characterization of a new alternative 5' UTR end of ClC-5 mRNA in human renal tissue and leukocytes.
Forino, Monica; Graziotto, Romina; Tosetto, Enrica; Gambaro, Giovanni; D'Angelo, Angela; Anglani, Franca
Mutations in the CLCN5 gene have been detected in Dent's disease and its phenotypic variants (X-linked recessive nephrolithiasis, X-linked recessive hypophosphatemic rickets, and idiopathic low-molecular-weight proteinuria of Japanese children). Dent's disease is a tubular disorder characterized by low-molecular-weight proteinuria, and nephrolithiasis associated with nephrocalcinosis and hypercalciuria. ClC-5 is the first chloride channel for which a definitive role in the trafficking and acidification-dependent recycling of apical membrane proteins has been established. In the course of CLCN5 SSCP analysis in patients with hypercalciuric nephrolithiasis, we detected a novel mutation at intron 2 of the CLCN5 gene, a T-to-G substitution, located 17 bp upstream of the AG acceptor site. To determine the effect of IVS2-17 T>G mutation on the correct splicing of intron 2, we studied ClC-5 transcripts in a patient's peripheral blood leukocytes by means of quantitative comparative RT/PCR, and found a new ClC-5 5' UTR isoform characterized by the untranslated exon 1b and by retention of intron 1b. This new isoform--isoform B1--was not correlated with mutation since it was detected also in control leukocytes and in renal tissues of kidney donors, thus confirming its physiological role. By RACE analysis we determined the putative transcriptional start site which is located at intron 1a, 251 nt upstream of the first nucleotide of the untranslated exon 1b. ORF analysis revealed that intron 1b retention in isoform B1 stabilizes the initiation of translation to the AGT at position 297 of the ClC-5 cDNA coding region. PMID:14673707
A functional alternative splicing mutation in AIRE gene causes autoimmune polyendocrine syndrome type 1.
Zhang, Junyu; Liu, Hongbin; Liu, Zhiyuan; Liao, Yong; Guo, Luo; Wang, Honglian; He, Lin; Zhang, Xiaodong; Xing, Qinghe
Autoimmune polyendocrine syndrome type 1 (APS-1) is a rare autosomal recessive disease defined by the presence of two of the three conditions: mucocutaneous candidiasis, hypoparathyroidism, and Addison's disease. Loss-of-function mutations of the autoimmune regulator (AIRE) gene have been linked to APS-1. Here we report mutational analysis and functional characterization of an AIRE mutation in a consanguineous Chinese family with APS-1. All exons of the AIRE gene and adjacent exon-intron sequences were amplified by PCR and subsequently sequenced. We identified a homozygous missense AIRE mutation c.463G>A (p.Gly155Ser) in two siblings with different clinical features of APS-1. In silico splice-site prediction and minigene analysis were carried out to study the potential pathological consequence. Minigene splicing analysis and subsequent cDNA sequencing revealed that the AIRE mutation potentially compromised the recognition of the splice donor of intron 3, causing alternative pre-mRNA splicing by intron 3 retention. Furthermore, the aberrant AIRE transcript was identified in a heterozygous carrier of the c.463G>A mutation. The aberrant intron 3-retaining transcript generated a truncated protein (p.G155fsX203) containing the first 154 AIRE amino acids and followed by 48 aberrant amino acids. Therefore, our study represents the first functional characterization of the alternatively spliced AIRE mutation that may explain the pathogenetic role in APS-1. PMID:23342054
Versatility of retroauricular mastoid donor site: a convenient valuable warehouse of various free graft tissues in cosmetic and reconstructive surgery.
Cho, Jeong Mok; Jeong, Jae Hoon; Woo, Kevin Volt; Lee, Yoon Ho
Soft-tissue deficiency is a critical issue in facial cosmetic and reconstructive surgery. Harvesting autografts from other anatomical sites has been a common practice in overcoming soft-tissue insufficiency for many years. However, donor-site complications and visible scars are of important concerns. Therefore, we would like to introduce an alternative donor site of free-tissue grafts and its inherent advantages: the retroauricular mastoid area located along the mastoid hair line. From August 1991 to June 2011, we performed facial reconstructive surgeries for cosmetic correction of disfigurements from both congenital and complications of previous cosmetic procedures on a total of 213 patients. These patients had undergone either 1 or more facial cosmetic surgeries in the past. In this study, our primary goal focused on revising facial asymmetries or defects from previous surgical scars, tissue contraction, undercorrection, or underdevelopment. For autograft harvesting, we incised an elliptical shape along the retroauricular hairline. We then harvested sufficient amount of skin, dermal fat, fascia, or a piece of the mastoid bone if needed. After harvesting, we closed the incisional area and covered it with a compressive dressing. In evaluation of our results, we compared the preoperative photographs with postoperative and constructed a survey on patient satisfaction. Overall, the patients in this study were greatly satisfied with their surgical results. No major complications were reported. As a result of our long-term study, we believe that the retroauricular mastoid area has been shown to be an indispensable donor site for a variety of autograft tissues in terms of safety, convenience, and versatility of its unique structural composition consisting of skin, dermal fat, fascia, and bone. PMID:24036825
Lysyl oxidase-like 4 is alternatively spliced in an anatomic site-specific manner in tumors involving the serosal cavities
Shulamit Sebban; Ben Davidson; Reuven Reich
Lysyl oxidase-like enzymes (LOXL) are expressed in various cancers. We analyzed the expression of LOXL2, LOXL3, and LOXL4\\u000a in cancers involving the serosal cavities�breast carcinoma, ovarian carcinoma, and malignant mesothelioma using reverse-transcriptase\\u000a polymerase chain reaction. We discovered two new alternative splice variants of LOXL4. The spliced segments were exon 9 (splice\\u000a variant 1) or both exons 8 and 9 (splice
Splicing factor SRSF1 negatively regulates alternative splicing of MDM2 under damage.
Comiskey, Daniel F; Jacob, Aishwarya G; Singh, Ravi K; Tapia-Santos, Aixa S; Chandler, Dawn S
Genotoxic stress induces alternative splicing of the oncogene MDM2 generating MDM2-ALT1, an isoform attributed with tumorigenic properties. However, the mechanisms underlying this event remain unclear. Here we explore MDM2 splicing regulation by utilizing a novel minigene that mimics endogenous MDM2 splicing in response to UV and cisplatinum-induced DNA damage. We report that exon 11 is necessary and sufficient for the damage-specific alternative splicing of the MDM2 minigene and that the splicing factor SRSF1 binds exon 11 at evolutionarily conserved sites. Interestingly, mutations disrupting this interaction proved sufficient to abolish the stress-induced alternative splicing of the MDM2 minigene. Furthermore, SRSF1 overexpression promoted exclusion of exon 11, while its siRNA-mediated knockdown prevented the stress-induced alternative splicing of endogenous MDM2. Additionally, we observed elevated SRSF1 levels under stress and in tumors correlating with the expression of MDM2-ALT1. Notably, we demonstrate that MDM2-ALT1 splicing can be blocked by targeting SRSF1 sites on exon 11 using antisense oligonucleotides. These results present conclusive evidence supporting a negative role for SRSF1 in MDM2 alternative splicing. Importantly, we define for the first time, a clear-cut mechanism for the regulation of damage-induced MDM2 splicing and present potential strategies for manipulating MDM2 expression via splicing modulation. PMID:25845590
Splicing factor SRSF1 negatively regulates alternative splicing of MDM2 under damage
Comiskey, Daniel F.; Jacob, Aishwarya G.; Singh, Ravi K.; Tapia-Santos, Aixa S.; Chandler, Dawn S.
Genotoxic stress induces alternative splicing of the oncogene MDM2 generating MDM2-ALT1, an isoform attributed with tumorigenic properties. However, the mechanisms underlying this event remain unclear. Here we explore MDM2 splicing regulation by utilizing a novel minigene that mimics endogenous MDM2 splicing in response to UV and cisplatinum-induced DNA damage. We report that exon 11 is necessary and sufficient for the damage-specific alternative splicing of the MDM2 minigene and that the splicing factor SRSF1 binds exon 11 at evolutionarily conserved sites. Interestingly, mutations disrupting this interaction proved sufficient to abolish the stress-induced alternative splicing of the MDM2 minigene. Furthermore, SRSF1 overexpression promoted exclusion of exon 11, while its siRNA-mediated knockdown prevented the stress-induced alternative splicing of endogenous MDM2. Additionally, we observed elevated SRSF1 levels under stress and in tumors correlating with the expression of MDM2-ALT1. Notably, we demonstrate that MDM2-ALT1 splicing can be blocked by targeting SRSF1 sites on exon 11 using antisense oligonucleotides. These results present conclusive evidence supporting a negative role for SRSF1 in MDM2 alternative splicing. Importantly, we define for the first time, a clear-cut mechanism for the regulation of damage-induced MDM2 splicing and present potential strategies for manipulating MDM2 expression via splicing modulation. PMID:25845590
DNA donors for sequencing at Celera, Craig VenterSite: DNA Interactive (www.dnai.org)
NSDL National Science Digital Library
Interviewee: Craig Venter DNAi Location:Genome>the project>players>private project The private project's DNA donors Craig Venter, the leader of the private genome effort at Celera Genomics, talks about the sources of the DNA used in their sequence.
A novel HLA-B*39 allele (HLA-B*3916) due to a rare mutation causing cryptic splice site activation.
Tamouza, R; El Kassar, N; Schaeffer, V; Carbonnelle, E; Tatari, Z; Marzais, F; Fortier, C; Poirier, J C; Sadki, K; Bernaudin, F; Toubert, A; Krishnamoorthy, R; Charron, D
A novel HLA-B*39 variant, found in an African patient with sickle cell anemia undergoing bone marrow transplantation is described. Initially suspected by inconsistent serological typing (B-blank, Bw6), then recognized by PCR-SSP, and finally characterized by nucleotide sequencing, this novel allele is designated HLA-B*3916. It differs from HLA-B*3910 by a point mutation (G to C) at position 17 of exon 3 causing glutamine to histidine change at codon 96 of alpha(2) domain, a conserved position among HLA class I alleles. cDNA sequence analysis further revealed the presence of both normally and abnormally spliced mRNA species in established cell lines. The abnormal species correspond to partial truncation of exon 3 presumably due to the nucleotide change in exon 3, which constitutes a new consensus acceptor splice site within this exon. We postulate that the observed blank is essentially the consequence of qualitative change in a critical region of this novel antigen as abnormal mRNA species are relatively less abundant than normal species. Because the residue 96 of the HLA class I heavy chain is directly involved in interaction with alpha(2)m, another interesting possibility is that an aminoacid change in this position would perturb such interaction and consequently could affect the serological specificity of B*3916, or its expression or both. PMID:10773349
Skin Thickness of the Anterior, Anteromedial, and Anterolateral Thigh: A Cadaveric Study for Split-Skin Graft Donor Sites
Ward, John; Quondamatteo, Fabio; Dockery, Peter; Kelly, John L
Background The depth of graft harvest and the residual dermis available for reepithelization primarily influence the healing of split-skin graft donor sites. When the thigh region is chosen, the authors hypothesize based on thickness measurements that the anterolateral region is the optimal donor site. Methods Full-thickness skin specimens were sampled from the anteromedial, anterior, and anterolateral regions of human cadavers. Skin specimens were cut perpendicularly with a custom-made precision apparatus to avoid the overestimation of thickness measurements. The combined epidermal and dermal thicknesses (overall skin thickness) were measured using a digital calliper. The specimens were histologically stained to visualize their basement membrane, and microscopy images were captured. Since the epidermal thickness varies across the specimen, a stereological method was used to eliminate observer bias. Results Epidermal thickness represented 2.5% to 9.9% of the overall skin thickness. There was a significant difference in epidermal thickness from one region to another (P<0.05). The anterolateral thigh region had the most consistent and highest mean epidermal thickness (60�3.2 �m). We observed that overall skin thickness increased laterally from the anteromedial region to the anterior and anterolateral regions of the thigh. The overall skin thickness measured 1,032�435 �m in the anteromedial region compared to 1,220�257 �m in the anterolateral region. Conclusions Based on skin thickness measurements, the anterolateral thigh had the thickest epidermal and dermal layers. We suggest that the anterolateral thigh region is the optimal donor site for split-skin graft harvests from the thigh. PMID:25396179
Autosomal recessive deafness 1A (DFNB1A) in Yakut population isolate in Eastern Siberia: extensive accumulation of the splice site mutation IVS1+1G>A in GJB2 gene as a result of founder effect.
Barashkov, Nikolay A; Dzhemileva, Lilya U; Fedorova, Sardana A; Teryutin, Fedor M; Posukh, Olga L; Fedotova, Elvira E; Lobov, Simeon L; Khusnutdinova, Elza K
Hereditary forms of hearing impairment (HI) caused by GJB2 (Cx26) mutations are the frequent sensory disorders registered among newborns in various human populations. In this study, we present data on the molecular, audiological and population features of autosomal recessive deafness 1A (DFNB1A) associated with the donor splicing site IVS1+1G>A mutation of GJB2 gene in Yakut population isolate of the Sakha Republic (Yakutia) located in Eastern Siberia (Russian Federation). The Yakut population exhibits high frequency of some Mendelian disorders, which are rare in other populations worldwide. Mutational analysis of GJB2 gene in 86 unrelated Yakut patients with congenital HI without other clinical features has been performed. In this study, we registered a large cohort of Yakut patients homozygous for the IVS1+1G>A mutation (70 unrelated deaf subjects in total). Detailed audiological analysis of 40 deaf subjects with genotype IVS1+1G>A/IVS1+1G>A revealed significant association of this genotype with mostly symmetrical bilateral severe to profound HI (85% severe-to-profound HI versus 15% mild-to-moderate HI, P<0.05). The highest among six investigated Eastern Siberian populations carrier frequency of the IVS1+1G>A mutation (11.7%) has been found in Yakut population. Reconstruction of 140 haplotypes with IVS1+1G>A mutation demonstrates the common origin of all mutant chromosomes found in Yakuts. The age of mutation was estimated to be approximately 800 years. These findings characterize Eastern Siberia as the region with the most extensive accumulation of the IVS1+1G>A mutation in the world as a result of founder effect. PMID:21776002
Autosomal recessive axonal form of Charcot-Marie-Tooth Disease caused by compound heterozygous 3'-splice site and Ser130Cys mutation in the GDAP1 gene.
Kabzi?ska, D; Kocha?ski, A; Drac, H; Ryniewicz, B; Rowi?ska-Marci?ska, K; Hausmanowa-Petrusewicz, I
A recessive demyelinating subtype of Charcot-Marie-Tooth disease called CMT4 is a heterogeneous group of disorders. A relatively frequent form of recessive CMT (CMT4 A) has been mapped to the chromosome 8 q21 and shown to be caused by mutations in the ganglioside-induced differentiation protein 1 (GDAP1) gene. Twenty mutations in the GDAP1 gene have been reported in patients suffering from the axonal and demyelinating forms of CMT disease. In this study we report two novel mutations in the GDAP1 gene in a patient suffering from CMT2 disease and whose parents were asymptomatic carriers of a Ser130Cys and 3'-splice site (311-1G > A) mutation, respectively. PMID:15944907
The evolution of an alternatively spliced exon in the alphaA-crystallin gene.
van Dijk, M A; Sweers, M A; de Jong, W W
The evolutionary aspects of alternative splicing, as a mechanism to increase the diversity of gene products, are poorly understood. Here we analyse the evolution of a 69-bp exon that is alternatively spliced in the primary transcript of the gene for the mammalian eye lens protein alphaA-crystallin. In rodents, the skipping of this exon 2 is attributed to the presence of a non-consensus 5' splice site GC, and results in the expression of 10-20% of alphaA(ins)-crystallin, with an insert of 23 residues, as compared with normal alphaA-crystallin. alphaA(ins)-crystallin is also expressed in some non-rodent mammals, including kangaroo, while lacking in others. We now demonstrate that the alternatively spliced exon 2 is present in mammals from different orders that do not express alphaA(ins)-crystallin. The expression of this exon has thus been silenced independently in various lineages. Sequence comparison in 16 species reveals that--whether or not alphaA(ins)-crystallin is expressed--exon 2 is always flanked by the non-consensus donor splice site GC, while a consensus branch point sequence and 3' pyrimidine-rich region are hardly detectable in the downstream intron. Increased numbers of amino acid replacements in the peptide encoded by exon 2 indicate that it is subject to much lower selective constraints than the exons that code for normal alphaA-crystallin. The absence of any apparent advantage at the protein level may suggest that exon 2 DNA sequences are conserved as cis-acting factors for proper splicing of the alphaA-crystallin transcript. PMID:11443354
Regulation of Alternative Splicing in Vivo by Overexpression of Antagonistic Splicing Factors
NASA Astrophysics Data System (ADS)
Caceres, Javier F.; Stamm, Stefan; Helfman, David M.; Krainer, Adrian R.
The opposing effects of SF2/ASF and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 influence alternative splicing in vitro. SF2/ASF or hnRNP A1 complementary DNAs were transiently overexpressed in HeLa cells, and the effect on alternative splicing of several cotransfected reporter genes was measured. Increased expression of SF2/ASF activated proximal 5' splice sites, promoted inclusion of a neuron-specific exon, and prevented abnormal exon skipping. Increased expression of hnRNP A1 activated distal 5' splice sites. Therefore, variations in the intracellular levels of antagonistic splicing factors influence different modes of alternative splicing in vivo and may be a natural mechanism for tissue-specific or developmental regulation of gene expression.
Prelaminating the fascial radial forearm flap by using tissue-engineered mucosa: improvement of donor and recipient sites.
Lauer, G; Schimming, R; Gellrich, N C; Schmelzeisen, R
In reconstructive surgery, prelamination of free flaps using split-thickness skin is an established technique to avoid the creation of a considerable defect at the donor site, for example, in the case of a radial forearm flap. For oral and maxillofacial surgery, this technique is less than optimal for the recipient site because the transferred skin is inadequate to form a lining in the oral cavity. To create mucosa-lined free flaps, prelamination using pieces of split-thickness mucosa has been performed. However, the availability of donor sites for harvesting mucosa is limited. The present study combines a tissue-engineering technique with free flap surgery to create mucosa-lined flaps with the intention of improving the tissue quality at the recipient site and decreasing donor-site morbidity. On five patients undergoing resection of squamous cell carcinoma of the oral cavity, the radial forearm flap was prelaminated with a tissue-engineered mucosa graft to reconstruct intraoral defects. Using 10 x 5 mm biopsies of healthy mucosa, keratinocytes were cultured for 12 days and seeded onto collagen membranes (4.5 x 9 cm). After 3 days, the mucosal keratinocyte collagen membrane was implanted subcutaneously at the left or right lower forearm to prelaminate the fascial radial forearm flap. One week later, resection of the squamous cell carcinoma was performed, and the free fascial radial forearm flap pre- laminated with tissue-engineered mucosa was transplanted into the defect and was microvascularly anastomosed. Resection defects up to a size of 5 x 8 cm were covered. In four patients, the graft healed without complications. In one patient, an abscess developed in the resection cavity without jeopardizing the flap. During the postoperative healing period, the membrane detached and a vulnerable pale-pink, glassy hyperproliferative wound surface was observed. This surface developed into normal-appearing healthy mucosa after 3 to 4 weeks. In the postoperative follow-up period, such functions as mouth opening and closing and speech attested to the success of the tissue-engineering technique for flap prelamination. PMID:11711928
Radiographic evaluation of the symphysis menti as a donor site for an autologous bone graft in pre-implant surgery
Di Bari, Roberto; Coronelli, Roberto
Purpose This study was performed to obtain a quantitative evaluation of the cortical and cancellous bone graft harvestable from the mental and canine regions, and to evaluate the cortical vestibular thickness. Materials and Methods This study collected cone-beam computed tomographic (CBCT) images of 100 Italian patients. The limits of the mental region were established: 5 mm in front of the medial margin of each mental foramen, 5 mm under the apex of each tooth present, and above the inferior mandibular cortex. Cortical and cancellous bone volumes were evaluated using SimPlant software (SimPlant 3-D Pro, Materialize, Leuven, Belgium) tools. In addition, the cortical vestibular thickness (minimal and maximal values) was evaluated in 3 cross-sections corresponding to the right canine tooth (3R), the median section (M), and the left canine tooth (3L). Results The cortical volume was 0.71�0.23 mL (0.27-1.96 mL) and the cancellous volume was 2.16�0.76 mL (0.86-6.28 mL). The minimal cortical vestibular thickness was 1.54�0.41 mm (0.61-3.25 mm), and the maximal cortical vestibular thickness was 3.14�0.75mm(1.01-5.83 mm). Conclusion The use of the imaging software allowed a patient-specific assessment of mental and canine region bone availability. The proposed evaluation method might help the surgeon in the selection of the donor site by the comparison between bone availability in the donor site and the reconstructive exigency of the recipient site. PMID:24083206
An Aberrant Splice Acceptor Site Due to a Novel Intronic Nucleotide Substitution in MSX1 Gene Is the Cause of Congenital Tooth Agenesis in a Japanese Family
Tatematsu, Tadashi; Kimura, Masashi; Nakashima, Mitsuko; Machida, Junichiro; Yamaguchi, Seishi; Shibata, Akio; Goto, Hiroki; Nakayama, Atsuo; Higashi, Yujiro; Miyachi, Hitoshi; Shimozato, Kazuo; Matsumoto, Naomichi; Tokita, Yoshihito
Congenital tooth agenesis is caused by mutations in the MSX1, PAX9, WNT10A, or AXIN2 genes. Here, we report a Japanese family with nonsyndromic tooth agenesis caused by a novel nucleotide substitution in the intronic region between exons 1 and 2 of the MSX1 gene. Because the mutation is located 9?bp before exon 2 (c.452-9G>A), we speculated that the nucleotide substitution would generate an abnormal splice site. Using cDNA analysis of an immortalized patient blood cell, we confirmed that an additional 7-nucleotide sequence was inserted at the splice junction between exons 1 and 2 (c.451_452insCCCTCAG). The consequent frameshift generated a homeodomain-truncated MSX1 (p.R151fsX20). We then studied the subcellular localization of truncated MSX1 protein in COS cells, and observed that it had a whole cell distribution more than a nuclear localization, compared to that of wild-type protein. This result suggests a deletion of the nuclear localization signal, which is mapped to the MSX1 homeodomain. These results indicate that this novel intronic nucleotide substitution is the cause of tooth agenesis in this family. To date, most MSX1 variants isolated from patients with tooth agenesis involve single amino acid substitutions in the highly conserved homeodomain or deletion mutants caused by frameshift or nonsense mutations. We here report a rare case of an intronic mutation of the MSX1 gene responsible for human tooth agenesis. In addition, the missing tooth patterns were slightly but significantly different between an affected monozygotic twin pair of this family, showing that epigenetic or environmental factors also affect the phenotypic variations of missing teeth among patients with nonsyndromic tooth agenesis caused by an MSX1 haploinsufficiency. PMID:26030286
Differential splicing of human androgen receptor pre-mRNA in X-linked reifenstein syndrome, because of a deletion involving a putative branch site
Ris-Stalpers, C.; Verleun-Mooijman, M.C.T.; Blaeij, T.J.P. de; Brinkmann, A.O.; Degenhart, H.J.; Trapman, J. (Erasmus Univ., Rotterdam (Netherlands))
The analysis of the androgen receptor (AR) gene, mRNA, and protein in a subject with X-linked Reifenstein syndrome (partial androgen insensitivity) is reported. The presence of two mature AR transcripts in genital skin fibroblasts of the patient is established, and, by reverse transcriptase-PCR and RNase transcription analysis, the wild-type transcript and a transcript in which exon 3 sequences are absent without disruption of the translational reading frame are identified. Sequencing and hybridization analysis show a deletion of >6 kb in intron 2 of the human AR gene, starting 18 bp upstream of exon 3. The deletion includes the putative branch-point sequence (BPS) but not the acceptor splice site on the intron 2/exon 3 boundary. The deletion of the putative intron 2 BPS results in 90% inhibition of wild-type splicing. The mutant transcript encodes an AR protein lacking the second zinc finger of the DNA-binding domain. Western/immunoblotting analysis is used to show that the mutant AR protein is expressed in genital skin fibroblasts of the patient. The residual 10% wild-type transcript can be the result of the use of a cryptic BPS located 63 bp upstream of the intron 2/exon 3 boundary of the mutant AR gene. The mutated AR protein has no transcription-activating potential and does not influence the transactivating properties of the wild-type AR, as tested in cotransfection studies. It is concluded that the partial androgen-insensitivity syndrome of this patient is the consequence of the limited amount of wild-type AR protein expressed in androgen target cells, resulting from the deletion of the intron 2 putative BPS. 42 refs., 6 figs., 1 tab.
Species-specific difference in expression and splice-site choice in Inpp5b, an inositol polyphosphate 5-phosphatase paralogous to the enzyme deficient in Lowe Syndrome.
Bothwell, Susan P; Farber, Leslie W; Hoagland, Adam; Nussbaum, Robert L
The oculocerebrorenal syndrome of Lowe (OCRL; MIM #309000) is an X-linked human disorder characterized by congenital cataracts, mental retardation, and renal proximal tubular dysfunction caused by loss-of-function mutations in the OCRL gene that encodes Ocrl, a type II phosphatidylinositol bisphosphate (PtdIns4,5P(2)) 5-phosphatase. In contrast, mice with complete loss-of-function of the highly homologous ortholog Ocrl have no detectable renal, ophthalmological, or central nervous system abnormalities. We inferred that the disparate phenotype between Ocrl-deficient humans and mice was likely due to differences in how the two species compensate for loss of the Ocrl enzyme. We therefore turned our attention to Inpp5b, another type II PtdIns4,5P(2) 5-phosphatase encoded by Inpp5b in mice and INPP5B in humans, as potential compensating genes in the two species, because Inpp5b/INPP5B are the most highly conserved paralogs to Ocrl/OCRL in the respective genomes of both species and Inpp5b demonstrates functional overlap with Ocrl in mice in vivo. We used in silico sequence analysis, reverse-transcription PCR, quantitative PCR, and transient transfection assays of promoter function to define splice-site usage and the function of an internal promoter in mouse Inpp5b versus human INPP5B. We found mouse Inpp5b and human INPP5B differ in their transcription, splicing, and primary amino acid sequence. These observations form the foundation for analyzing the functional basis for the difference in how Inpp5b and INPP5B compensate for loss of Ocrl function and, by providing insight into the cellular roles of Ocrl and Inpp5b, aid in the development of a model system in which to study Lowe syndrome. PMID:20872266
SAW: A Method to Identify Splicing Events from RNA-Seq Data Based on Splicing Fingerprints
Ning, Kang; Fermin, Damian
Splicing event identification is one of the most important issues in the comprehensive analysis of transcription profile. Recent development of next-generation sequencing technology has generated an extensive profile of alternative splicing. However, while many of these splicing events are between exons that are relatively close on genome sequences, reads generated by RNA-Seq are not limited to alternative splicing between close exons but occur in virtually all splicing events. In this work, a novel method, SAW, was proposed for the identification of all splicing events based on short reads from RNA-Seq. It was observed that short reads not in known gene models are actually absent words from known gene sequences. An efficient method to filter and cluster these short reads by fingerprint fragments of splicing events without aligning short reads to genome sequences was developed. Additionally, the possible splicing sites were also determined without alignment against genome sequences. A consensus sequence was then generated for each short read cluster, which was then aligned to the genome sequences. Results demonstrated that this method could identify more than 90% of the known splicing events with a very low false discovery rate, as well as accurately identify, a number of novel splicing events between distant exons. PMID:20706591
Introns and alternative splicing in choanoflagellates
intron �definition. �Journal �of �Cell �Biology, �2008. �definition �in � Schizosaccharomyces �pombe. �Molecular �and �Cellular �Biology, �definition �may �facilitate �splice � site �selection �in �RNAs �with �multiple �exons. �Molecular �and �Cellular �Biology, �
Conservation and Sex-Specific Splicing of the transformer Gene in the Calliphorids Cochliomyia hominivorax, Cochliomyia macellaria and Lucilia sericata
Li, Fang; Vensko, Steven P.; Belikoff, Esther J.; Scott, Maxwell J.
Transformer (TRA) promotes female development in several dipteran species including the Australian sheep blowfly Lucilia cuprina, the Mediterranean fruit fly, housefly and Drosophila melanogaster. tra transcripts are sex-specifically spliced such that only the female form encodes full length functional protein. The presence of six predicted TRA/TRA2 binding sites in the sex-specific female intron of the L. cuprina gene suggested that tra splicing is auto-regulated as in medfly and housefly. With the aim of identifying conserved motifs that may play a role in tra sex-specific splicing, here we have isolated and characterized the tra gene from three additional blowfly species, L. sericata, Cochliomyia hominivorax and C. macellaria. The blowfly adult male and female transcripts differ in the choice of splice donor site in the first intron, with males using a site downstream of the site used in females. The tra genes all contain a single TRA/TRA2 site in the male exon and a cluster of four to five sites in the male intron. However, overall the sex-specific intron sequences are poorly conserved in closely related blowflies. The most conserved regions are around the exon/intron junctions, the 3? end of the intron and near the cluster of TRA/TRA2 sites. We propose a model for sex specific regulation of tra splicing that incorporates the conserved features identified in this study. In L. sericata embryos, the male tra transcript was first detected at around the time of cellular blastoderm formation. RNAi experiments showed that tra is required for female development in L. sericata and C. macellaria. The isolation of the tra gene from the New World screwworm fly C. hominivorax, a major livestock pest, will facilitate the development of a �male-only� strain for genetic control programs. PMID:23409170
The public Human Genome Project's DNA donors, Eric LanderSite: DNA Interactive (www.dnai.org)
NSDL National Science Digital Library
Interviewee: Eric Lander DNAi Location: Genome>The Project>players>Public consortium The public's DNA donors Eric Lander, director of the Whitehead Institute Center for Genome Research, explains where the DNA donors for the first reference sequence came from.
[Extra-abdominal desmoid tumor in a scar of donor-site of a latissimus dorsi flap: case report].
Paradol, P-O; Toussoun, G; Delbaere, M; Delaporte, T; Delay, E
Tumefaction arising lately after latissimus dorsi flap harvest are rare and observed in 1 or 2% of the cases. These lesions are frequently related to kystic sero-hematoma and are easily and efficiently treated with surgical excision. In some rare circumstances, a tumoral evolution can mimic a kystic sero-hematoma. We will discuss one case of desmoid tumor arising from a latissimus dorsi flap donor-site scar. The subject was a 45 years old woman who had a breast reconstruction following mastectomy. A dorsal tumefaction, with a benign aspect, was observed during the follow-up period. The biopsy showed an extra-abdominal desmoid tumor. The patient was treated with a large excision of the lesion and reconstructed using two opposing local cutaneous advancing flaps. No radicalization was necessary. No sign of recurrence has been observed at 4 years follow-up. PMID:17418929
A Contracted DNA Repeat in LHX3 Intron 5 Is Associated with Aberrant Splicing and Pituitary Dwarfism in German Shepherd Dogs
Voorbij, Annemarie M. W. Y.; van Steenbeek, Frank G.; Vos-Loohuis, Manon; Martens, Ellen E. C. P.; Hanson-Nilsson, Jeanette M.; van Oost, Bernard A.; Kooistra, Hans S.; Leegwater, Peter A.
Dwarfism in German shepherd dogs is due to combined pituitary hormone deficiency of unknown genetic cause. We localized the recessively inherited defect by a genome wide approach to a region on chromosome 9 with a lod score of 9.8. The region contains LHX3, which codes for a transcription factor essential for pituitary development. Dwarfs have a deletion of one of six 7 bp repeats in intron 5 of LHX3, reducing the intron size to 68 bp. One dwarf was compound heterozygous for the deletion and an insertion of an asparagine residue in the DNA-binding homeodomain of LHX3, suggesting involvement of the gene in the disorder. An exon trapping assay indicated that the shortened intron is not spliced efficiently, probably because it is too small. We applied bisulfite conversion of cytosine to uracil in RNA followed by RT-PCR to analyze the splicing products. The aberrantly spliced RNA molecules resulted from either skipping of exon 5 or retention of intron 5. The same splicing defects were observed in cDNA derived from the pituitary of dwarfs. A survey of similarly mutated introns suggests that there is a minimal distance requirement between the splice donor and branch site of 50 nucleotides. In conclusion, a contraction of a DNA repeat in intron 5 of canine LHX3 leads to deficient splicing and is associated with pituitary dwarfism. PMID:22132174
MYC regulates the core pre-mRNA splicing machinery as an essential step in lymphomagenesis.
Koh, Cheryl M; Bezzi, Marco; Low, Diana H P; Ang, Wei Xia; Teo, Shun Xie; Gay, Florence P H; Al-Haddawi, Muthafar; Tan, Soo Yong; Osato, Motomi; Sab�, Arianna; Amati, Bruno; Wee, Keng Boon; Guccione, Ernesto
Deregulated expression of the MYC transcription factor occurs in most human cancers and correlates with high proliferation, reprogrammed cellular metabolism and poor prognosis. Overexpressed MYC binds to virtually all active promoters within a cell, although with different binding affinities, and modulates the expression of distinct subsets of genes. However, the critical effectors of MYC in tumorigenesis remain largely unknown. Here we show that during lymphomagenesis in E�-myc transgenic mice, MYC directly upregulates the transcription of the core small nuclear ribonucleoprotein particle assembly genes, including Prmt5, an arginine methyltransferase that methylates Sm proteins. This coordinated regulatory effect is critical for the core biogenesis of small nuclear ribonucleoprotein particles, effective pre-messenger-RNA splicing, cell survival and proliferation. Our results demonstrate that MYC maintains the splicing fidelity of exons with a weak 5' donor site. Additionally, we identify pre-messenger-RNAs that are particularly sensitive to the perturbation of the MYC-PRMT5 axis, resulting in either intron retention (for example, Dvl1) or exon skipping (for example, Atr, Ep400). Using antisense oligonucleotides, we demonstrate the contribution of these splicing defects to the anti-proliferative/apoptotic phenotype observed in PRMT5-depleted E�-myc B cells. We conclude that, in addition to its well-documented oncogenic functions in transcription and translation, MYC also safeguards proper pre-messenger-RNA splicing as an essential step in lymphomagenesis. PMID:25970242
Effects of a silicone-coated polyamide net dressing and calcium alginate on the healing of split skin graft donor sites: a prospective randomised trial.
O'Donoghue, J M; O'Sullivan, S T; O'Shaughnessy, M; O'Connor, T P
An open randomised prospectively controlled trial was performed to assess the healing efficacy, slippage rate and degree of discomfort on removal of calcium alginate and a silicone-coated polyamide net dressing on split skin graft donor sites. Sixteen patients were randomised to the calcium alginate group and 14 to the silicone-coated group. The donor sites were assessed at days 7, 10, 14 and up to day 21. The mean time to healing in the calcium alginate group was 8.75 +/- 0.78 days (range 7 to 14 days) compared to 12 +/- 0.62 days (range 7 to 16 days) for the silicone-coated group (p < 0.01). Although more silicone-coated dressings slipped (5 versus 1), the difference was not statistically significant. Pain during the first dressing change was assessed using a visual analogue pain scale. Although no significant differences were found between the groups, it was necessary to change the dressing protocol in the silicone-coated arm of the trial after entering the first two patients. Overlaid absorbent gauze adhered to the donor site through the fenestrations in the dressing necessitating the placement of paraffin gauze between the experimental dressing and the overlying cotton gauze. There was one infection in the study, occurring in the alginate group. Based on these results we recommend calcium alginate as the dressing of choice for split skin graft donor sites. PMID:10815307
Alternative splicing generates a second isoform of the catalytic A subunit of the vacuolar H(+)-ATPase.
Hernando, N; Bartkiewicz, M; Collin-Osdoby, P; Osdoby, P; Baron, R
We have identified a second isoform of the catalytic A subunit of the vacuolar H+ pump in chicken osteoclasts. In this isoform (A2) a 72-bp cassette replaces a 90-bp cassette present in the classical A1 isoform. The A1-specific cassette encodes a region of the protein that contains one of the three ATP-binding consensus sequences (the P-loop) identified in this polypeptide, as well as the pharmacologically relevant Cys254. In contrast, the A2-specific cassette does not contain any of these features. These two isoforms, which appear to be ubiquitously expressed, are encoded by a single gene and are generated by alternative splicing of two mutually exclusive exons. The alternative RNA processing involves the recognition of a single site, the boundary between the A2- and A1-specific exons, as either acceptor (in A1) or donor (in A2) splice site. Images Fig. 2 Fig. 3 Fig. 4 PMID:7597085
Tissue-specific splicing mutation in acute intermittent porphyria
Grandchamp, B.; Picat, C. (Laboratoire de Genetique Moleculaire, Paris (France)); Mignotte, V.; Romeo, P.H.; Goossens, M. (Institut National de la Sante et de la Recherche Medicale, Creteil (France)); Wilson, J.H.P.; Sandkuyl, L. (Erasmus Univ., Rotterdam (Netherlands)); Te Velde, K. (Saint Geertruiden Hospital, Deventer (Netherlands)); Nordmann, Y. (Hopital Louis Mourier, Colombes (France))
An inherited deficiency of porphobilinogen deaminase in humans is responsible for the autosomal dominant disease acute intermittent porphyria. Different classes of mutations have been described at the protein level suggesting that this is a heterogeneous disease. It was previously demonstrated that porphobilinogen deaminase is encoded by two distinct mRNA species expressed in a tissue-specific manner. Analysis of the genomic sequences indicated that these two mRNAs are transcribed from two promoters and only differ in their first exon. The first mutation identified in the human porphobilinogen deaminase gene is a single-base substitution (G {yields} A) in the canonical 5{prime} splice donor site of intron 1. This mutation leads to a particular subtype of acute intermittent porphyria characterized by the restriction of the enzymatic defect to nonerythropoietic tissues. Hybridization analysis using olignonucleotide probes after in vitro amplification of genomic DNA offers another possibility of detecting asymptomatic carriers of the mutation in affected families.
Molecular basis for type 1 antithrombin deficiency: identification of two novel point mutations and evidence for a de novo splice site mutation.
Jochmans, K; Lissens, W; Yin, T; Michiels, J J; van der Luit, L; Peerlinck, K; De Waele, M; Liebaers, I
Inherited type 1 antithrombin (AT) deficiency is characterized by a reduction in both immunologically and functionally detectable protein. The disorder is associated with a high risk of thromboembolic disease. We have investigated the molecular basis of type 1 AT deficiency in three unrelated families. We have used the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis, followed by direct sequencing of the seven exons and the intron-exon junctions of the AT gene. Two novel point mutations were identified. A T to C single-base substitution was found in codon 421 in exon 6 (nucleotide position 13380), leading to an AT 421 isoleucine to threonine substitution. In another kindred, one of three Cs at nucleotide (nt) positions 5448 to 5450 in exon 3A (codon 151 or 152) was deleted, resulting in a frameshift mutation and predicting premature termination of protein translation at codon 251. In a third family, a previously reported G to A substitution, at nt position 9788 in intron 4, 14 bp in front of exon 5, was found. We have demonstrated the creation of a de novo exon 5 splice site by ectopic transcript analysis of lymphocyte mRNA. In all cases, the affected individuals were heterozygous for the mutation and no variant AT protein was detected. PMID:7949130
Nuclear proteins that bind the pre-mRNA 3' splice site sequence r(UUAG/G) and the human telomeric DNA sequence d(TTAGGG)n.
Ishikawa, F; Matunis, M J; Dreyfuss, G; Cech, T R
HeLa cell nuclear proteins that bind to single-stranded d(TTAGGG)n, the human telomeric DNA repeat, were identified and purified by a gel retardation assay. Immunological data and peptide sequencing experiments indicated that the purified proteins were identical or closely related to the heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, A2-B1, D, and E and to nucleolin. These proteins bound to RNA oligonucleotides having r(UUAGGG) repeats more tightly than to DNA of the same sequence. The binding was sequence specific, as point mutation of any of the first 4 bases [r(UUAG)] abolished it. The fraction containing D and E hnRNPs was shown to bind specifically to a synthetic oligoribonucleotide having the 3' splice site sequence of the human beta-globin intervening sequence 1, which includes the sequence UUAGG. Proteins in this fraction were further identified by two-dimensional gel electrophoresis as D01, D02, D1*, and E0; intriguingly, these members of the hnRNP D and E groups are nuclear proteins that are not stably associated with hnRNP complexes. These studies establish the binding specificities of these D and E hnRNPs. Furthermore, they suggest the possibility that these hnRNPs could perhaps bind to chromosome telomeres, in addition to having a role in pre-mRNA metabolism. Images PMID:8321232
Discovery of candidate disease genes in ENU-induced mouse mutants by large-scale sequencing, including a splice-site mutation in nucleoredoxin.
Boles, Melissa K; Wilkinson, Bonney M; Wilming, Laurens G; Liu, Bin; Probst, Frank J; Harrow, Jennifer; Grafham, Darren; Hentges, Kathryn E; Woodward, Lanette P; Maxwell, Andrea; Mitchell, Karen; Risley, Michael D; Johnson, Randy; Hirschi, Karen; Lupski, James R; Funato, Yosuke; Miki, Hiroaki; Marin-Garcia, Pablo; Matthews, Lucy; Coffey, Alison J; Parker, Anne; Hubbard, Tim J; Rogers, Jane; Bradley, Allan; Adams, David J; Justice, Monica J
An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU) mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn), inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing. PMID:20011118
A Prospective, Randomized, Placebo Controlled, Double Blind Study of Silicone Gel in Prevention of Hypertrophic Scar at Donor Site of Skin Grafting
Chittoria, Ravi Kumar; Padi, Tirupathi Rao
Background: Hypertrophic scarring at donor site of skin grafting is prevalent among Asians. The effectiveness of silicone gel in scar prevention may influence the surgeons and patients regarding its routine use during the postoperative period. Aims and Objectives: To study the efficacy of silicone gel in prevention of hypertrophic scars at donor site of skin grafting. Design: Prospective randomized placebo controlled double blind study. Setting: The study was conducted in the department of Plastic Surgery, Sri Venkateswara Institute of Medical Sciences (SVIMS) University, Tirupati, Andhra Pradesh, India from June 2007 to June 2009. Patients were recruited during follow-up in the OPD. Materials and Methods: The susceptibility to scar development varied among patients; therefore, donor site scars were divided into upper half and lower half. Two types of coded gel prepared by an independent pharmacist were used on either half. Thus, selection and assessment biases and confounders were eliminated. Results: 100 scars in 50 patients were randomized into two arms, 50 control and 50 silicone gel. The median age was 25.5 years and there were 30 men (60%) and 20 women (40%). Thirty-seven patients (74%) had good compliance. The overall incidence of donor site hypertrophic scar was 94% (47 out of 50). At the second month postoperatively, the silicone gel scars were scored lower when compared with the control scars. The differences were statistically significant in all parameters, including pigmentation (P = 0.001), Vascularity (P = 0.010), pliability (P = 0.001), and height (P = 0.010). Conclusion: The effect of silicone gel in prevention of hypertrophic scar development in donor site scars is promising. Success of silicone gel in its prophylactic role will create its routine use in all types of surgery to minimize the formation of hypertrophic scars in the early postoperative period. PMID:23723598
Species-specific difference in expression and splice-site choice in Inpp5b, an inositol polyphosphate 5-phosphatase paralogous to the enzyme deficient in Lowe Syndrome
Bothwell, Susan P.; Farber, Leslie W.; Hoagland, Adam
The oculocerebrorenal syndrome of Lowe (OCRL; MIM #309000) is an X-linked human disorder characterized by congenital cataracts, mental retardation, and renal proximal tubular dysfunction caused by loss-of-function mutations in the OCRL gene that encodes Ocrl, a type II phosphatidylinositol bisphosphate (PtdIns4,5P2) 5-phosphatase. In contrast, mice with complete loss-of-function of the highly homologous ortholog Ocrl have no detectable renal, ophthalmological, or central nervous system abnormalities. We inferred that the disparate phenotype between Ocrl-deficient humans and mice was likely due to differences in how the two species compensate for loss of the Ocrl enzyme. We therefore turned our attention to Inpp5b, another type II PtdIns4,5P2 5-phosphatase encoded by Inpp5b in mice and INPP5B in humans, as potential compensating genes in the two species, because Inpp5b/INPP5B are the most highly conserved paralogs to Ocrl/OCRL in the respective genomes of both species and Inpp5b demonstrates functional overlap with Ocrl in mice in vivo. We used in silico sequence analysis, reverse-transcription PCR, quantitative PCR, and transient transfection assays of promoter function to define splice-site usage and the function of an internal promoter in mouse Inpp5b versus human INPP5B. We found mouse Inpp5b and human INPP5B differ in their transcription, splicing, and primary amino acid sequence. These observations form the foundation for analyzing the functional basis for the difference in how Inpp5b and INPP5B compensate for loss of Ocrl function and, by providing insight into the cellular roles of Ocrl and Inpp5b, aid in the development of a model system in which to study Lowe syndrome. Electronic supplementary material The online version of this article (doi:10.1007/s00335-010-9281-7) contains supplementary material, which is available to authorized users. PMID:20872266
Mutations in the Caenorhabditis elegans U2AF Large Subunit UAF-1 Alter the Choice of a 3' Splice Site In Vivo
Ma, Long
The removal of introns from eukaryotic RNA transcripts requires the activities of five multi-component ribonucleoprotein complexes and numerous associated proteins. The lack of mutations affecting splicing factors essential ...
Mutations in the Caenorhabditis elegans U2AF Large Subunit UAF-1 Alter the Choice of a 3? Splice Site In Vivo
Long Ma; H. Robert Horvitz
The removal of introns from eukaryotic RNA transcripts requires the activities of five multi-component ribonucleoprotein complexes and numerous associated proteins. The lack of mutations affecting splicing factors essential for animal survival has limited the study of the in vivo regulation of splicing. From a screen for suppressors of the Caenorhabditis elegans unc-93(e1500) rubberband Unc phenotype, we identified mutations in genes
A comparison of the donor-site morbidity after using the prelaminated fasciomucosal flap and the fasciocutaneous radial forearm flap for intraoral reconstruction.
Nehrer-Tairych, G V; Millesi, W; Schuhfried, O; Rath, T
Since 1996 we have performed mucosal prelamination of the distal radial forearm flap for functional reconstruction of defects of the intraoral lining. This study was undertaken to demonstrate that the prelaminated fasciomucosal radial forearm flap can provide physiological oropharyngeal reconstruction with mucus-producing tissue, while avoiding the donor-site complications of the fasciocutaneous radial forearm flap. We examined the donor hand at least 6 months postoperatively in 20 patients after using a prelaminated fasciomucosal radial forearm flap and in 15 patients after harvesting a classical fasciocutaneous radial forearm flap. The evaluation of hand function included range of motion, grip power, pinch power, sensibility and vascular analysis in both hands. The patients were asked about cold intolerance, pain and any restrictions in daily life, and the cosmetic appearance of the donor hand was noted. In the prelaminated-flap group (n 20), two patients had decreased wrist extension, and one of these patients also had reduced strength and mild hypaesthesia in the donor hand. In the classical-fasciocutaneous-flap group (n 15), six patients had decreased wrist extension, five patients had reduced strength and four patients had diminished sensibility in the donor hand. Painful neuromas were found only after fasciocutaneous flaps (three cases). Subjective assessment revealed restricted hand function in one patient in the prelaminated-flap group, and in five patients who had undergone fasciocutaneous flap transfer. The results of this study show that using the prelaminated fasciomucosal radial forearm flap minimises the donor-site morbidity. Furthermore, we were able to improve the cosmetic appearance of this very exposed region. PMID:12041971
Management of split-thickness donor sites with synthetic wound dressings: results of a comparative clinical study.
Markl, Peter; Prantl, Lukas; Schreml, Stephan; Babilas, Philipp; Landthaler, Michael; Schwarze, Hardy
This prospective, randomized, single-blinded, clinical study aimed at evaluating 3 different synthetic wound dressings for treating split-thickness skin graft donor sites. Seventy-seven patients were randomly assigned to 3 study groups: Suprathel, Biatain-Ibu, Mepitel. Wounds were inspected daily until complete reepithelization. Ease of care, treatment costs, and scar development after a 6 months follow-up were evaluated. Suprathel showed significant (P ? 0.001) pain reduction after 24 hours but increasing pain scores on the 5th day of treatment. Biatain-Ibu showed significant pain relief immediately after application and during the entire treatment period (P < 0.05). Mepitel did not show any significant pain reduction. No differences were seen with regard to healing time, quality of reepithelization, and scar development. Biatain-Ibu had the lowest overall treatment costs (P ? 0.001). The investigated materials did not differ with regard to quality and acceleration of the healing process, but Biatain-Ibu seems to be the most appropriate dressing material in terms of cost-effectiveness. PMID:20841998
EASI�enrichment of alternatively spliced isoforms
Venables, Julian P.; Burn, John
Alternative splicing produces more than one protein from the majority of genes and the rarer forms can have dominant functions. Instability of alternative transcripts can also hinder the study of regulation of gene expression by alternative splicing. To investigate the true extent of alternative splicing we have developed a simple method of enriching alternatively spliced isoforms (EASI) from PCRs using beads charged with Thermus aquaticus single-stranded DNA-binding protein (T.Aq ssb). This directly purifies the single-stranded regions of heteroduplexes between alternative splices formed in the PCR, enabling direct sequencing of all the rare alternative splice forms of any gene. As a proof of principle the alternative transcripts of three tumour suppressor genes, TP53, MLH1 and MSH2, were isolated from testis cDNA. These contain missing exons, cryptic splice sites or include completely novel exons. EASI beads are stable for months in the fridge and can be easily combined with standard protocols to speed the cloning of novel transcripts. PMID:16951290
Splicing of human immunodeficiency virus RNA is position-dependent suggesting sequential removal of introns from the 50 end
Jens Bohne; Harald Wodrich; Hans-Georg Krausslich
Transcription of the HIV-1 genome yields a single primary transcript, which is alternatively spliced to .30 mRNAs. Productive infection depends on inef- ficient and regulated splicing and appears to pro- ceed in a tight 50 to 30 order. To analyse whether sequential splicing is mediated by the quality of splice sites or by the position of an intron, we inserted
Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF
Chen, Zhiqi; Ma, Xuezhong; Zhang, Jianhua; Hu, Jim; Gorczynski, Reginald M.
CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200tr) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200. PMID:20558599
ASD: the Alternative Splicing Database
Thangavel Alphonse Thanaraj; Stefan Stamm; Francis Clark; Jean-jack M. Riethoven; Vincent Le Texier; Juha Muilu
Alternative splicing is widespread in mammalian gene expression, and variant splice patterns are often specific to different stages of development, particular tissues or a disease state. There is a need to systematically collect data on alternatively spliced exons, introns and splice isoforms, and to annotate this data. The Alternative Splicing Database consortium has been addressing this need, and is committed
Genetic analysis of autosomal recessive osteopetrosis in Chuvashiya: the unique splice site mutation in TCIRG1 gene spread by the founder effect
Bliznetz, Elena A; Tverskaya, Svetlana M; Zinchenko, Rena A; Abrukova, Anna V; Savaskina, Ekaterina N; Nikulin, Maxim V; Kirillov, Alexander G; Ginter, Evgeny K; Polyakov, Alexander V
The rare malignant disorder autosomal recessive osteopetrosis (OPTB) is one of the most prevalent autosomal recessive diseases in the Chuvash Republic of Russia. The purpose of this study was to determine the underlying molecular cause of osteopetrosis in Chuvashiya and to reveal the factors causing the unusual high frequency of the disease in this region. Having assumed a founder effect, we performed linkage disequilibrium (LD) mapping of the OPTB locus at the TCIRG1 region and found a unique splice site mutation c.807+5G>A in all Chuvashian OPTB patients studied. We then analyzed the mutational change in mRNA and detected an intron insertion within the mutant transcript, resulting in a frameshift and premature stop-codon formation (p.Leu271AspfsX231). A decreased expression of the mutant transcript was also detected, which may have been the result of nonsense-mediated decay. Real-time qPCR and MLPA� melting curve analysis-based systems were designed and used for c.807+5G>A mutation screening. In addition to analyzing the gene frequency in Chuvashiya, we also estimated three other populations in the Volga-Ural region (Mari, Udmurt and Bashkir). We found a 1.68% prevalence in Chuvashiya (calculated disease frequency, 1/3500 newborns) and a 0.84% in the Mari population (1/14?000 newborns). The haplotype analysis revealed that all OPTB cases in Chuvashians and Marians originated from a single mutational event and the age of the mutation in Chuvashians was estimated to be approximately 890 years. PMID:19172990
A short interspersed repetitive element provides a new 3' acceptor site for trans-splicing in certain ribosomal P2 beta protein genes of Trypanosoma cruzi.
Vazquez, M P; Schijman, A G; Levin, M J
Four Trypanosoma cruzi genomic DNA fragments carrying different TcP2 beta genes have been isolated and sequenced. Three of them had a single TcP2 beta gene, while the 3.8-kb-long DNA segment encoding the TcP2 beta-H1.8 locus showed two TcP2 beta genes arranged in tandem. These genes were physically connected by a 428-bp-long DNA sequence that was also located immediately 5' to the first gene and immediately 3' to the second. Comparison of the 4 TcP2 beta gene loci, suggested that the insertion of this repeated element originated the duplication of its target sequence, a poly(dT) stretch. Approximately 1200 copies of this short sequence, named short interspersed repetitive element (SIRE), were found scattered in the genome. Analysis of the 5' non-coding regions of different TcP2 beta mRNAs, and RNA-PCR experiments suggested that the insertion of a SIRE upstream of a TcP2 beta-H1.8 gene introduced a new 3' spliced leader (SL) acceptor site in the TcP2 beta-H1.8 pre-mRNAs, encoded within the SIRE. Consequently, in the mature H1.8 mRNA the SL sequence is followed by 38 bases directly transcribed from the SIRE. Structural and functional features of this repeated element reveal similarity to the short interspersed repetitive DNA sequences detected in the genomes of several microorganisms. PMID:7935610
Proximity-dependent and proximity-independent trans-splicing in mammalian cells
Viles, Kristi D.; Sullenger, Bruce A.
Most human pre-mRNAs are cis-spliced, removing introns and joining flanking exons of the same RNA molecule. However, splicing of exons present on separate pre-mRNA molecules can also occur. This trans-splicing reaction can be exploited by pre-trans-splicing molecules (PTMs), which are incapable of cis-splicing. PTM-mediated trans-splicing has been utilized to repair mutant RNAs as a novel approach to gene therapy. Herein we explore how the site of PTM expression influences trans-splicing activity. We stably inserted a PTM expression cassette into the genome of HEK293 cells, generating clonal lines with single, unique insertion sites. We analyzed trans-splicing to the gene where the PTM was integrated, as well as genes neighboring these loci. We observed some pre-mRNAs only serve as substrates for trans-splicing when they are expressed in immediate proximity to the PTM expression site. The need for PTMs to be in close proximity with pre-mRNAs to trans-splice with them is consistent with the observation that pre-mRNA cis-splicing occurs cotranscriptionally. Interestingly, we identified several cellular pre-mRNAs in one localized area that serve as trans-splicing substrates irrespective of the PTM expression site. Thus, we find multiple cellular pre-mRNAs require PTM expression in close proximity to trans-splice while others do not. PMID:18441053
Translational control of intron splicing in eukaryotes.
Jaillon, Olivier; Bouhouche, Khaled; Gout, Jean-Fran�ois; Aury, Jean-Marc; Noel, Benjamin; Saudemont, Baptiste; Nowacki, Mariusz; Serrano, Vincent; Porcel, Betina M; S�gurens, B�atrice; Le Mou�l, Anne; Lep�re, Gersende; Sch�chter, Vincent; B�termier, Mireille; Cohen, Jean; Wincker, Patrick; Sperling, Linda; Duret, Laurent; Meyer, Eric
Most eukaryotic genes are interrupted by non-coding introns that must be accurately removed from pre-messenger RNAs to produce translatable mRNAs. Splicing is guided locally by short conserved sequences, but genes typically contain many potential splice sites, and the mechanisms specifying the correct sites remain poorly understood. In most organisms, short introns recognized by the intron definition mechanism cannot be efficiently predicted solely on the basis of sequence motifs. In multicellular eukaryotes, long introns are recognized through exon definition and most genes produce multiple mRNA variants through alternative splicing. The nonsense-mediated mRNA decay (NMD) pathway may further shape the observed sets of variants by selectively degrading those containing premature termination codons, which are frequently produced in mammals. Here we show that the tiny introns of the ciliate Paramecium tetraurelia are under strong selective pressure to cause premature termination of mRNA translation in the event of intron retention, and that the same bias is observed among the short introns of plants, fungi and animals. By knocking down the two P. tetraurelia genes encoding UPF1, a protein that is crucial in NMD, we show that the intrinsic efficiency of splicing varies widely among introns and that NMD activity can significantly reduce the fraction of unspliced mRNAs. The results suggest that, independently of alternative splicing, species with large intron numbers universally rely on NMD to compensate for suboptimal splicing efficiency and accuracy. PMID:18202663
The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: Causes and consequences
Michael Krawczak; Jochen Reiss; David N. Cooper
A total of 101 different examples of point mutations, which lie in the vicinity of mRNA splice junctions, and which have been held to be responsible for a human genetic disease by altering the accuracy of efficiency of mRNA splicing, have been collated. These data comprise 62 mutations at 5' splice sites, 26 at 3' splice sites and 13 that
Control of protein splicing by intein fragment reassembly.
Southworth, M W; Adam, E; Panne, D; Byer, R; Kautz, R; Perler, F B
Inteins are protein splicing elements that mediate their excision from precursor proteins and the joining of the flanking protein sequences (exteins). In this study, protein splicing was controlled by splitting precursor proteins within the Psp Pol-1 intein and expressing the resultant fragments in separate hosts. Reconstitution of an active intein was achieved by in vitro assembly of precursor fragments. Both splicing and intein endonuclease activity were restored. Complementary fragments from two of the three fragmentation positions tested were able to splice in vitro. Fragments resulting in redundant overlaps of intein sequences or containing affinity tags at the fragmentation sites were able to splice. Fragment pairs resulting in a gap in the intein sequence failed to splice or cleave. However, similar deletions in unfragmented precursors also failed to splice or cleave. Single splice junction cleavage was not observed with single fragments. In vitro splicing of intein fragments under native conditions was achieved using mini exteins. Trans-splicing allows differential modification of defined regions of a protein prior to extein ligation, generating partially labeled proteins for NMR analysis or enabling the study of the effects of any type of protein modification on a limited region of a protein. PMID:9463370
Highlights of Alternative Splicing Regulation Session: Yes, No, Maybe--A History of Paradigm Shifts
NSDL National Science Digital Library
Thomas A. Cooper (Baylor College of Medicine; Departments of Pathology and Molecular and Cellular Biology REV)
Cooper summarizes the discussions and presentations from the session entitled "Control of Splice Site Selection" held at the Sixth Annual Meeting of the RNA Society. Paradigms are shifting as experiments show that some of the proteins involved in regulating splicing can act as splicing enhancers or repressors, depending on the cellular context. The complex interactions among the ribonucleoproteins (RNPs) and proteins, and the role of cis elements, in controlling cell-specific splicing are highlighted. The importance of properly regulated splicing is emphasized by examples of disease pathologies in which alternative splicing is aberrant.
Analysis of Aberrantly Spliced Transcripts of a Novel de novo GNAS Mutant in a Male with Albright Hereditary Osteodystrophy and PHP1A.
Ham, H-J; Baek, K-H; Lee, J-Y; Kim, S Y; Mo, E Y; Kim, E S; Han, J H; Moon, S-D
Pseudohypoparathyroidism (PHP) is a genetic disorder due to target-organ unresponsiveness to parathyroid hormone (PTH). PHP type 1A (PHP1A) is an autosomal dominant disease characterized by Albright hereditary osteodystrophy (AHO) and PTH resistance caused by defects at the GNAS locus. We analyzed the GNAS gene in a male with typical AHO and elevated PTH levels. We identified a novel de novo heterozygous mutation at the splice donor site in intron-7 (IVS7+1G>A, c.585+1G>A) of the GNAS gene. No GNAS mutations were detected in his parents. Our patient was diagnosed with PHP1A due to a heterozygous de novo mutation in the GNAS gene. Reverse transcriptase (RT) PCR analysis and sequencing revealed that this de novo splice mutation generated alternative splicing errors leading to the formation of 2 mutant transcripts: one with exon-7 deleted, the other with whole intron-7 included. To investigate whether these aberrantly spliced transcripts were stable, we assessed the differential expression of GNAS mRNAs in the proband's blood by real-time quantitative RT-PCR. In the proband, the relative expression levels of wild-type, exon-7-deleted, and intron-7-included GNAS mRNAs were 0.21, 6.12E-07, and 1.08E-04, respectively, relative to wild-type GNAS mRNA from a healthy control (set at 1.0). This suggests that this novel de novo splicing mutation generates rapidly decaying mutant transcripts, which might affect stimulatory G-protein activity and give rise to this sporadic case. In conclusion, this is an interesting report of aberrantly spliced mRNAs from a de novo splice mutation of the GNAS gene causing PHP1A in a male. PMID:25502941
Variation in alternative splicing across human tissues
Yeo, Gene; Holste, Dirk; Kreiman, Gabriel; Burge, Christopher B
Background Alternative pre-mRNA splicing (AS) is widely used by higher eukaryotes to generate different protein isoforms in specific cell or tissue types. To compare AS events across human tissues, we analyzed the splicing patterns of genomically aligned expressed sequence tags (ESTs) derived from libraries of cDNAs from different tissues. Results Controlling for differences in EST coverage among tissues, we found that the brain and testis had the highest levels of exon skipping. The most pronounced differences between tissues were seen for the frequencies of alternative 3' splice site and alternative 5' splice site usage, which were about 50 to 100% higher in the liver than in any other human tissue studied. Quantifying differences in splice junction usage, the brain, pancreas, liver and the peripheral nervous system had the most distinctive patterns of AS. Analysis of available microarray expression data showed that the liver had the most divergent pattern of expression of serine-arginine protein and heterogeneous ribonucleoprotein genes compared to the other human tissues studied, possibly contributing to the unusually high frequency of alternative splice site usage seen in liver. Sequence motifs enriched in alternative exons in genes expressed in the brain, testis and liver suggest specific splicing factors that may be important in AS regulation in these tissues. Conclusions This study distinguishes the human brain, testis and liver as having unusually high levels of AS, highlights differences in the types of AS occurring commonly in different tissues, and identifies candidate cis-regulatory elements and trans-acting factors likely to have important roles in tissue-specific AS in human cells. PMID:15461793
A{sup -2} {yields} G transition at the 3{prime} acceptor splice site of IVS17 characterizes the COL2A1 gene mutation in the original Stickler syndrome kindred
Williams, C.J.; Ganguly, A.; Considine, E. [Thomas Jefferson Univ., Philadelphia, PA (United States)] [and others] [Thomas Jefferson Univ., Philadelphia, PA (United States); and others
Hereditary progressive arthro-ophthalmopathy, or {open_quotes}Stickler syndrome,{close_quotes} is an autosomal dominant osteochondrodysplasia characterized by a variety of ocular and skeletal anomalies which frequently lead to retinal detachment and precocious osteoarthritis. A variety of mutations in the COL2A1 gene have been identified in {open_quotes}Stickler{close_quotes} families; in most cases studied thus far, the consequence of mutation is the premature generation of a stop codon. We report here the characterization of a COL2A1 gene mutation in the original kindred described by Stickler et al. Conformational sensitive gel electrophoresis (CSGE) was used to screen for mutations in the entire COL2A1 gene in an affected member from the kindred. A prominent heteroduplex species was noted in the polymerase chain reaction (PCR) product from a region of the gene including exons 17 to 20. Direct sequencing of PCR-amplified genomic DNA resulted in the identification of a base substitution at the A{sup -2} position of the 3{prime} splice acceptor site of IVS17. Sequencing of DNA from affected and unaffected family members confirmed that the mutation segregated with the disease phenotype. Reverse transcriptase-PCR analysis of poly A+ RNA demonstrated that the mutant allele utilized a cryptic splice site in exon 18 of the gene, eliminating 16 bp at the start of exon 18. This frameshift eventually results in a premature termination codon. These findings are the first report of a splice site mutation in classical Stickler syndrome and they provide a satisfying historical context in which to view COL2A1 mutations in this dysplasia. 25 refs., 3 figs., 1 tab.
Separating some splicing models
Paola Bonizzoni; Claudio Ferretti; Giancarlo Mauri; Rosalba Zizza
This work shows that the family of languages generated by splicing as defined in the early paper by T. Head is strictly included in the family defined by later de nitions by Head, Gh. Paun, and others. It also proves the strict inclusion of this latter family in the splicing family defined by D. Pixton when proving the regularity of
Interplay between DMD Point Mutations and Splicing Signals in Dystrophinopathy Phenotypes
Juan-Mateu, Jon�s; Gonz�lez-Quereda, Lidia; Rodr�guez, Maria Jos�; Verdura, Edgard; L�zaro, Kira; Jou, Cristina; Nascimento, Andr�s; Jim�nez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jes�s; Baiget, Montserrat; Gallano, Pia
DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements. PMID:23536893
Laparoendoscopic single-site (LESS) vs laparoscopic living-donor nephrectomy: a systematic review and meta-analysis.
Autorino, Riccardo; Brandao, Luis Felipe; Sankari, Bashir; Zargar, Homayoun; Laydner, Humberto; Ak�a, Oktay; De Sio, Marco; Mirone, Vincenzo; Chueh, Shih-Chieh J; Kaouk, Jihad H
The aim of this study was to provide a systematic review and meta-analysis of reports comparing laparoendoscopic single-site (LESS) living-donor nephrectomy (LDN) vs standard laparoscopic LDN (LLDN). A systematic review of the literature was performed in September 2013 using PubMed, Scopus, Ovid and The Cochrane library databases. Article selection proceeded according to the search strategy based on Preferred Reporting Items for Systematic Reviews and Meta-analyses criteria. Weighted mean differences (WMDs) were used to measure continuous variables and odds ratios (ORs) to measure categorical ones. Nine publications meeting eligibility criteria were identified, including 461 LESS LDN and 1006 LLDN cases. There were more left-side cases in the LESS LDN group (96.5% vs 88.6%, P < 0.001). Meta-analysis of extractable data showed that LLDN had a shorter operative time (WMD 15.06?min, 95% confidence interval [CI] 4.9-25.1; P = 0.003), without a significant difference in warm ischaemia time (WMD 0.41?min, 95% CI -0.02 to 0.84; P = 0.06). Estimated blood loss was lower for LESS LDN (WMD -22.09?mL, 95% CI -29.5 to -14.6; P < 0.001); however, this difference was not clinically significant. There was a greater likelihood of conversion for LESS LDN (OR 13.21, 95% CI 4.65-37.53; P < 0.001). Hospital stay was similar (WMD -0.11 days, 95% CI -0.33 to 0.12; P = 0.35), as well as the visual analogue pain score at discharge (WMD -0.31, 95% CI -0.96 to 0.35; P = 0.36), but the analgesic requirement was lower for LESS LDN (WMD -2.58?mg, 95% CI -5.01 to -0.15; P = 0.04). Moreover, there was no difference in the postoperative complication rate (OR 1.00, 95% CI 0.65-1.54; P = 0.99). Renal function of the recipient, as based on creatinine levels at 1 month, showed similar outcomes between groups (WMD 0.10?mg/dL, -0.09 to 0.29; P = 0.29). In conclusion, LESS LDN represents an emerging option for living kidney donation. This procedure offers comparable surgical and early functional outcomes to the conventional LLDN, with a lower analgesic requirement. However, it is more technically challenging than LLDN, as shown by a greater likelihood of conversion. The role of LESS LDN remains to be defined. PMID:24588876
Plant pre-mRNA splicing in fission yeast, Schizosaccharomyces pombe
Bhaskarjyoti Sarmah; Niranjan Chakraborty; Subhra Chakraborty; Asis Datta
Pre-mRNA splicing or the removal of introns from precursor messenger RNAs depends on the accurate recognition of intron sequences by the splicing machinery. We have analyzed various aspects of intron sequence and structure in relation to splice site selection and splicing efficiency of a plant gene AmA1 in Schizosaccharomyces pombe. Earlier, we reported the cloning of AmA1, a seed albumin
The splicing landscape is globally reprogrammed during male meiosis
Schmid, Ralf; Grellscheid, Sushma Nagaraja; Ehrmann, Ingrid; Dalgliesh, Caroline; Danilenko, Marina; Paronetto, Maria Paola; Pedrotti, Simona; Grellscheid, David; Dixon, Richard J.; Sette, Claudio; Eperon, Ian C.; Elliott, David J.
Meiosis requires conserved transcriptional changes, but it is not known whether there is a corresponding set of RNA splicing switches. Here, we used RNAseq of mouse testis to identify changes associated with the progression from mitotic spermatogonia to meiotic spermatocytes. We identified ?150 splicing switches, most of which affect conserved protein-coding exons. The expression of many key splicing regulators changed in the course of meiosis, including downregulation of polypyrimidine tract binding protein (PTBP1) and heterogeneous nuclear RNP A1, and upregulation of nPTB, Tra2?, muscleblind, CELF proteins, Sam68 and T-STAR. The sequences near the regulated exons were significantly enriched in target sites for PTB, Tra2? and STAR proteins. Reporter minigene experiments investigating representative exons in transfected cells showed that PTB binding sites were critical for splicing of a cassette exon in the Ralgps2 mRNA and a shift in alternative 5? splice site usage in the Bptf mRNA. We speculate that nPTB might functionally replace PTBP1 during meiosis for some target exons, with changes in the expression of other splicing factors helping to establish meiotic splicing patterns. Our data suggest that there are substantial changes in the determinants and patterns of alternative splicing in the mitotic-to-meiotic transition of the germ cell cycle. PMID:24038356
Topical phenytoin in the treatment of split-thickness skin autograft donor sites: a comparative study with polyurethane membrane drape and conventional dressing.
Yadav, J K; Singhvi, A M; Kumar, N; Garg, S
The effectiveness of topical phenytoin as a wound healing agent was compared with that of OpSite (Smith & Nephew) and a conventional topical antibiotic dressing (Soframycin, Roussel) in a controlled study of 60 patients with partial-thickness skin autograft donor sites on the lower extremities. Mean time to complete healing (complete epithelialization) was 6.2 +/- 1.6 days in the phenytoin-treated group (30 patients), compared to 8.6 +/- 2.2 days with OpSite (15 patients), and 12.6 +/- 3.4 days in the 15 Soframycin-treated patients. The differences between the treatment groups were significant at P < 0.001. Mean pain scores were also lower in the phenytoin-treated group, 0.40 +/- 0.55 vs. 0.66 +/- 0.60 with OpSite (P < 0.05) and 1.4 +/- 0.50 with the conventional dressing (P < 0.001). Both phenytoin and OpSite were superior to the Soframycin dressing with respect to bacterial contamination and wound infection as measured by Gram stains of wound smears, swab and aspirate (OpSite) cultures, and clinical assessments (P < 0.001) carried out on the fifth day of treatment. No local or systemic adverse effects of the three agents used were noted. Phenytoin appears to be an effective, low-cost and safe method for the treatment of partial-thickness skin graft donor sites, comparing very favourably with, and in some aspects superior to, occlusive dressings. Further clinical use and evaluation of topical phenytoin are merited. PMID:8357478
What Is the Ideal Free Flap for Soft Tissue Reconstruction? A Ten-Year Experience of Microsurgical Reconstruction Using 334 Latissimus Dorsi Flaps From a Universal Donor Site.
Kim, Jeong Tae; Kim, Sang Wha; Youn, Seungki; Kim, Youn Hwan
Microsurgical free tissue transfer is regarded as the best available method of tissue reconstruction for intractable defects. The ideal soft tissue flap is thought to be the anterolateral thigh flap. On the basis of 334 procedures involving the latissimus dorsi (LD) flap, we discuss the advantages of the LD flap over the current universal option, and we aimed to establish whether the LD could also gain universal status in all reconstructive fields.Three hundred thirty-four reconstructive procedures using the LD flap were performed in 322 patients between September 2002 and July 2012. In accordance with defect characteristics, we performed 334 procedures using flaps, which included the LD muscle flap with skin graft, the myocutaneous flap, the muscle-sparing flap, the perforator flap, the chimeric flap, and the 2-flap technique using the serratus anterior branch.Flap-related complications occurred in 21 patients (6.3%), including total and partial flap failure. In 253 cases, the donor site was closed primarily, and in the remaining cases, we used split-thickness skin grafts. Donor-site complications occurred in 20 cases (6%). In 11 of the 182 cases, no suitable perforators were identified during surgery.The advantages of the LD as a donor site include the possibility of various harvesting positions without position change, versatility of components, availability of muscle to fill extensive defects, and presence of thick fascia to enable full abdominal reconstruction. On the basis of our experience, we concluded that this flap has the potential to be used as widely as, or in preference to, the anterolateral thigh flap in most reconstructive areas. PMID:25785382
Identification of U2AF(35)-dependent exons by RNA-Seq reveals a link between 3' splice-site organization and activity of U2AF-related proteins.
Kralovicova, Jana; Knut, Marcin; Cross, Nicholas C P; Vorechovsky, Igor
The auxiliary factor of U2 small nuclear RNA (U2AF) is a heterodimer consisting of 65- and 35-kD proteins that bind the polypyrimidine tract (PPT) and AG dinucleotides at the 3' splice site (3'ss). The gene encoding U2AF35 (U2AF1) is alternatively spliced, giving rise to two isoforms U2AF35a and U2AF35b. Here, we knocked down U2AF35 and each isoform and characterized transcriptomes of HEK293 cells with varying U2AF35/U2AF65 and U2AF35a/b ratios. Depletion of both isoforms preferentially modified alternative RNA processing events without widespread failure to recognize 3'ss or constitutive exons. Over a third of differentially used exons were terminal, resulting largely from the use of known alternative polyadenylation (APA) sites. Intronic APA sites activated in depleted cultures were mostly proximal whereas tandem 3'UTR APA was biased toward distal sites. Exons upregulated in depleted cells were preceded by longer AG exclusion zones and PPTs than downregulated or control exons and were largely activated by PUF60 and repressed by CAPER?. The U2AF(35) repression and activation was associated with a significant interchange in the average probabilities to form single-stranded RNA in the optimal PPT and branch site locations and sequences further upstream. Although most differentially used exons were responsive to both U2AF subunits and their inclusion correlated with U2AF levels, a small number of transcripts exhibited distinct responses to U2AF35a and U2AF35b, supporting the existence of isoform-specific interactions. These results provide new insights into function of U2AF and U2AF35 in alternative RNA processing. PMID:25779042
Assembly of splicing complexes on exon 11 of the human insulin receptor gene does not correlate with splicing efficiency in-vitro
Webster, Nicholas JG; Evans, Lui-Guojing; Caples, Matt; Erker, Laura; Chew, Shern L
Background Incorporation of exon 11 of the insulin receptor gene is both developmentally and hormonally-regulated. Previously, we have shown the presence of enhancer and silencer elements that modulate the incorporation of the small 36-nucleotide exon. In this study, we investigated the role of inherent splice site strength in the alternative splicing decision and whether recognition of the splice sites is the major determinant of exon incorporation. Results We found that mutation of the flanking sub-optimal splice sites to consensus sequences caused the exon to be constitutively spliced in-vivo. These findings are consistent with the exon-definition model for splicing. In-vitro splicing of RNA templates containing exon 11 and portions of the upstream intron recapitulated the regulation seen in-vivo. Unexpectedly, we found that the splice sites are occupied and spliceosomal complex A was assembled on all templates in-vitro irrespective of splicing efficiency. Conclusion These findings demonstrate that the exon-definition model explains alternative splicing of exon 11 in the IR gene in-vivo but not in-vitro. The in-vitro results suggest that the regulation occurs at a later step in spliceosome assembly on this exon. PMID:15233842
Splicing enhancement in the yeast rp51b intron.
Libri, D; Lescure, A; Rosbash, M
Splicing enhancement in higher eukaryotes has been linked to SR proteins, to U1 snRNP, and to communication between splice sites across introns or exons mediated by protein-protein interactions. It has been previously shown that, in yeast, communication mediated by RNA-RNA interactions between the two ends of introns is a basis for splicing enhancement. We designed experiments of randomization-selection to isolate splicing enhancers that would work independently from RNA secondary structures. Surprisingly, one of the two families of sequences selected was essentially composed of 5' splice site variants. We show that this sequence enhances splicing independently of secondary structure, is exportable to heterologous contexts, and works in multiple copies with additive effects. The data argue in favor of an early role for splicing enhancement, possibly coincident with commitment complex formation. Genetic compensation experiments with U1 snRNA mutants suggest that U1 snRNP binding to noncanonical locations is required for splicing enhancement. PMID:10744020
Ratios of Four STAT3 Splice Variants in Human Eosinophils and Diffuse Large B Cell Lymphoma Cells.
Turton, Keren B; Annis, Douglas S; Rui, Lixin; Esnault, Stephane; Mosher, Deane F
Signal transducer and activator of transcription 3 (STAT3) is a key mediator of leukocyte differentiation and proliferation. The 3' end of STAT3 transcripts is subject to two alternative splicing events. One results in either full-length STAT3? or in STAT3?, which lacks part of the C-terminal transactivation domain. The other is at a tandem donor (5') splice site and results in the codon for Ser-701 being included (S) or excluded (?S). Despite the proximity of Ser-701 to the site of activating phosphorylation at Tyr-705, ?S/S splicing has barely been studied. Sequencing of cDNA from purified eosinophils revealed the presence of four transcripts (S-?, ?S-?, S-?, and ?S-?) rather than the three reported in publically available databases from which ?S-? is missing. To gain insight into regulation of the two alternative splicing events, we developed a quantitative(q) PCR protocol to compare transcript ratios in eosinophils in which STAT3 is upregulated by cytokines, activated B cell diffuse large B cell Lymphoma (DLBCL) cells in which STAT3 is dysregulated, and in germinal center B cell-like DLBCL cells in which it is not. With the exception of one line of activated B cell DLCBL cells, the four variants were found in roughly the same ratios despite differences in total levels of STAT3 transcripts. S-? was the most abundant, followed by S-?. ?S-? and ?S-? together comprised 15.6�4.0 % (mean�SD, n=21) of the total. The percentage of STAT3? variants that were ?S was 1.5-fold greater than of STAT3? variants that were ?S. Inspection of Illumina's "BodyMap" RNA-Seq database revealed that the ?S variant accounts for 10-26 % of STAT3 transcripts across 16 human tissues, with less variation than three other genes with the identical tandem donor splice site sequence. Thus, it seems likely that all cells contain the S-?, ?S-?, S-?, and ?S-? variants of STAT3. PMID:25984943
Ratios of Four STAT3 Splice Variants in Human Eosinophils and Diffuse Large B Cell Lymphoma Cells
Turton, Keren B.; Annis, Douglas S.; Rui, Lixin; Esnault, Stephane; Mosher, Deane F.
Signal transducer and activator of transcription 3 (STAT3) is a key mediator of leukocyte differentiation and proliferation. The 3' end of STAT3 transcripts is subject to two alternative splicing events. One results in either full-length STAT3? or in STAT3?, which lacks part of the C-terminal transactivation domain. The other is at a tandem donor (5') splice site and results in the codon for Ser-701 being included (S) or excluded (?S). Despite the proximity of Ser-701 to the site of activating phosphorylation at Tyr-705, ?S/S splicing has barely been studied. Sequencing of cDNA from purified eosinophils revealed the presence of four transcripts (S-?, ?S-?, S-?, and ?S-?) rather than the three reported in publically available databases from which ?S-? is missing. To gain insight into regulation of the two alternative splicing events, we developed a quantitative(q) PCR protocol to compare transcript ratios in eosinophils in which STAT3 is upregulated by cytokines, activated B cell diffuse large B cell Lymphoma (DLBCL) cells in which STAT3 is dysregulated, and in germinal center B cell-like DLBCL cells in which it is not. With the exception of one line of activated B cell DLCBL cells, the four variants were found in roughly the same ratios despite differences in total levels of STAT3 transcripts. S-? was the most abundant, followed by S-?. ?S-? and ?S-? together comprised 15.6�4.0 % (mean�SD, n=21) of the total. The percentage of STAT3? variants that were ?S was 1.5-fold greater than of STAT3? variants that were ?S. Inspection of Illumina�s �BodyMap� RNA-Seq database revealed that the ?S variant accounts for 10-26 % of STAT3 transcripts across 16 human tissues, with less variation than three other genes with the identical tandem donor splice site sequence. Thus, it seems likely that all cells contain the S-?, ?S-?, S-?, and ?S-? variants of STAT3. PMID:25984943
An intron 9 containing splice variant of PAX2
Busse, Antonia; Rietz, Anika; Schwartz, Stefan; Thiel, Eckhard; Keilholz, Ulrich
Background PAX2 is a transcription factor with an important role in embryogenic development. However, PAX2 expression was frequently identified in neoplasia responsible for the growth and survival of cancer cells. Due to alternative splicing of exon 6, exon 10 and exon 12 four isoforms of PAX2 are described so far. Methods The expression of an intron 9 containing PAX2 splice variant was analyzed in neoplastic B cell and solid tumor cell lines as well as in primary tumor samples by quantitative RT-PCR. PAX2 proteins were detected by Western Blot in a subset of cell lines. Results All 14 lymphoma cell lines expressed an undescribed PAX2 splice variant containing the entire intron 9 sequence and the exon 10 sequence. This splice variant could also be detected in 35 solid tumor cell lines, in leukemia and lymphoma as well as in colon carcinoma and melanoma patient samples and in blood samples of healthy donors. Expression of this new splice variant on protein level was verified by Western Blot analysis. Conclusion We discovered a previously undescribed intron 9 and exon 10 containing splice variant of PAX2 in B-cell neoplasia and in solid tumors on mRNA and protein level. PMID:19467152
Feasibility of Use of a Barbed Suture (V-Loc 180) for Quilting the Donor Site in Latissimus Dorsi Myocutaneous Flap Breast Reconstruction
Hussain, Tasadooq; Mahapatra, Tapan Kumar; McManus, Penelope Louise; Kneeshaw, Peter John
Background Latissimus dorsi (LD) myocutaneous flap is a popular method of breast reconstruction which can be associated with high incidence of seroma formation. Quilting sutures at the harvest site are used to reduce this. Barbed sutures are self anchoring sutures which avoid multiple knotting and can be useful in quilting. Methods A retrospective analysis of prospectively maintained database of patients who underwent LD flap breast reconstruction between January 2009 and January 2011 was carried out. Seroma formation at the harvest site, wound related complications, inpatient stay and duration of surgery were analysed and a comparison was made between two groups where quilting was done with barbed (V-Loc) suture and conventional polydioxanone (PDS) II sutures. Results Fifty-seven patients were included of which 33 had quilting by V-Loc sutures and in 24 patients PDS II suture was used. Median age in the PDS group was 55 years (interquartile range [IQR)], 45 to 61 years) which was comparable to the V-Loc group (53 years [IQR, 48 to 59 years]; P-value 0.948). Sixteen patients (28%) had significant seroma formation and 5 (9%) patients developed superficial wound dehiscence. Incidences of seroma or wound complications were comparable (P-value 0.378 and 1.00, respectively). Secondary outcomes such as total duration of surgery, total inpatient stay, total amount of drain at the donor site were also similar in two groups. Conclusions Use of barbed sutures for quilting the donor site in LD flap reconstruction is a feasible option and the associated seroma formation and wound complications are comparable with conventional sutures. PMID:23532830
Mechanisms and Regulation of Alternative Pre-mRNA Splicing.
Lee, Yeon; Rio, Donald C
Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms with limited gene numbers. Split eukaryotic genes contain intervening sequences or introns disrupting protein-coding exons, and intron removal occurs by repeated assembly of a large and highly dynamic ribonucleoprotein complex termed the spliceosome, which is composed of five small nuclear ribonucleoprotein particles, U1, U2, U4/U6, and U5. Biochemical studies over the past 10 years have allowed the isolation as well as compositional, functional, and structural analysis of splicing complexes at distinct stages along the spliceosome cycle. The average human gene contains eight exons and seven introns, producing an average of three or more alternatively spliced mRNA isoforms. Recent high-throughput sequencing studies indicate that 100% of human genes produce at least two alternative mRNA isoforms. Mechanisms of alternative splicing include RNA-protein interactions of splicing factors with regulatory sites termed silencers or enhancers, RNA-RNA base-pairing interactions, or chromatin-based effects that can change or determine splicing patterns. Disease-causing mutations can often occur in splice sites near intron borders or in exonic or intronic RNA regulatory silencer or enhancer elements, as well as in genes that encode splicing factors. Together, these studies provide mechanistic insights into how spliceosome assembly, dynamics, and catalysis occur; how alternative splicing is regulated and evolves; and how splicing can be disrupted by cis- and trans-acting mutations leading to disease states. These findings make the spliceosome an attractive new target for small-molecule, antisense, and genome-editing therapeutic interventions. PMID:25784052
WT1 interacts with the splicing protein RBM4 and regulates its ability to modulate alternative splicing in vivo
Markus, M. Andrea [Basic and Clinical Genomics Laboratory, School of Medical Sciences and Bosch Institute, Building F13, University of Sydney, NSW 2006 (Australia); Heinrich, Bettina [Institute of Biochemistry, University of Erlangen, 91054 Erlangen (Germany); Raitskin, Oleg [Department of Genetics, Hebrew University of Jerusalem, Jerusalem 91904 (Israel); Adams, David J. [Basic and Clinical Genomics Laboratory, School of Medical Sciences and Bosch Institute, Building F13, University of Sydney, NSW 2006 (Australia); Mangs, Helena [Basic and Clinical Genomics Laboratory, School of Medical Sciences and Bosch Institute, Building F13, University of Sydney, NSW 2006 (Australia); Goy, Christine [Basic and Clinical Genomics Laboratory, School of Medical Sciences and Bosch Institute, Building F13, University of Sydney, NSW 2006 (Australia); Ladomery, Michael [Centre for Research in Biomedicine, Bristol Genomics Research Institute, Faculty of Applied Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY (United Kingdom); Sperling, Ruth [Department of Genetics, Hebrew University of Jerusalem, Jerusalem 91904 (Israel); Stamm, Stefan [Institute of Biochemistry, University of Erlangen, 91054 Erlangen (Germany); Morris, Brian J. [Basic and Clinical Genomics Laboratory, School of Medical Sciences and Bosch Institute, Building F13, University of Sydney, NSW 2006 (Australia)]. E-mail: email@example.com
Wilm's tumor protein 1 (WT1), a protein implicated in various cancers and developmental disorders, consists of two major isoforms: WT1(-KTS), a transcription factor, and WT1(+KTS), a post-transcriptional regulator that binds to RNA and can interact with splicing components. Here we show that WT1 interacts with the novel splicing regulator RBM4. Each protein was found to colocalize in nuclear speckles and to cosediment with supraspliceosomes in glycerol gradients. RBM4 conferred dose-dependent and cell-specific regulation of alternative splicing of pre-mRNAs transcribed from several reporter genes. We found that overexpressed WT1(+KTS) abrogated this effect of RBM4 on splice-site selection, whereas WT1(-KTS) did not. We conclude that the (+KTS) form of WT1 is able to inhibit the effect of RBM4 on alternative splicing.
Genome-wide discovery of human splicing branchpoints.
Mercer, Tim R; Clark, Michael B; Andersen, Stacey B; Brunck, Marion E; Haerty, Wilfried; Crawford, Joanna; Taft, Ryan J; Nielsen, Lars K; Dinger, Marcel E; Mattick, John S
During the splicing reaction, the 5' intron end is joined to the branchpoint nucleotide, selecting the next exon to incorporate into the mature RNA and forming an intron lariat, which is excised. Despite a critical role in gene splicing, the locations and features of human splicing branchpoints are largely unknown. We use exoribonuclease digestion and targeted RNA-sequencing to enrich for sequences that traverse the lariat junction and, by split and inverted alignment, reveal the branchpoint. We identify 59,359 high-confidence human branchpoints in >10,000 genes, providing a first map of splicing branchpoints in the human genome. Branchpoints are predominantly adenosine, highly conserved, and closely distributed to the 3' splice site. Analysis of human branchpoints reveals numerous novel features, including distinct features of branchpoints for alternatively spliced exons and a family of conserved sequence motifs overlapping branchpoints we term B-boxes, which exhibit maximal nucleotide diversity while maintaining interactions with the keto-rich U2 snRNA. Different B-box motifs exhibit divergent usage in vertebrate lineages and associate with other splicing elements and distinct intron-exon architectures, suggesting integration within a broader regulatory splicing code. Lastly, although branchpoints are refractory to common mutational processes and genetic variation, mutations occurring at branchpoint nucleotides are enriched for disease associations. PMID:25561518
Pre-mRNA splicing of IgM exons M1 and M2 is directed by a juxtaposed splicing enhancer and inhibitor
Kan, Julie L.C.; Green, Michael R.
Splicing of certain pre-mRNA introns is dependent on an enhancer element, which is typically purine-rich. It is generally thought that enhancers increase the use of suboptimal splicing signals, and one specific proposal is that enhancers stabilize binding of U2AF65 to weak polypyrimidine (Py) tracts. Here, we test this model using an IgM pre-mRNA substrate, which contains a well-characterized enhancer. Although the enhancer was required for in vitro splicing, we found it had no effect on U2AF65 binding. Unexpectedly, replacement of the natural IgM Py tract, branchpoint, and 5? splice site with consensus splicing signals did not circumvent the enhancer requirement. These observations led us to identify a novel regulatory element within the IgM M2 exon that acts as a splicing inhibitor; removal of the inhibitor enabled splicing to occur in the absence of the enhancer. The IgM M2 splicing inhibitor is evolutionarily conserved, can inhibit the activity of an unrelated, constitutively spliced pre-mRNA, and acts by repressing splicing complex assembly. Interestingly, the inhibitor itself forms an ATP-dependent complex that contains U2 snRNP. We conclude that splicing of IgM exons M1 and M2 is directed by two juxtaposed regulatory elements�an enhancer and an inhibitor�and that a primary function of the enhancer is to counteract the inhibitor. PMID:10049361
Aberrant splicing in MLH1 and MSH2 due to exonic and intronic variants.
Pagenstecher, Constanze; Wehner, Maria; Friedl, Waltraut; Rahner, Nils; Aretz, Stefan; Friedrichs, Nicolaus; Sengteller, Marlies; Henn, Wolfram; Buettner, Reinhard; Propping, Peter; Mangold, Elisabeth
Single base substitutions in DNA mismatch repair genes which are predicted to lead either to missense or silent mutations, or to intronic variants outside the highly conserved splicing region are often found in hereditary nonpolyposis colorectal cancer (HNPCC) families. In order to use the variants for predictive testing in persons at risk, their pathogenicity has to be evaluated. There is growing evidence that some substitutions have a detrimental influence on splicing. We examined 19 unclassified variants (UVs) detected in MSH2 or MLH1 genes in patients suspected of HNPCC for expression at RNA level. We demonstrate that 10 of the 19 UVs analyzed affect splicing. For example, the substitution MLH1,c.2103G > C in the last position of exon 18 does not result in a missense mutation as theoretically predicted (p.Gln701His), but leads to a complete loss of exon 18. The substitution MLH1,c.1038G > C (predicted effect p.Gln346His) leads to complete inactivation of the mutant allele by skipping of exons 10 and 11, and by activation of a cryptic intronic splice site. Similarly, the intronic variant MLH1,c.306+2dupT results in loss of exon 3 and a frameshift mutation due to a new splice donor site 5 bp upstream. Furthermore, we confirmed complete exon skipping for the mutations MLH1,c.1731G > A and MLH1,c.677G > A. Partial exon skipping was demonstrated for the mutations MSH2,c.1275A > G, MLH1,c.588+5G > A, MLH1,c.790+4A > G and MLH1,c.1984A > C. In contrast, five missense mutations (MSH2,c.4G > A, MSH2,c.2123T > A, MLH1,c.464T > G, MLH1,c.875T > C and MLH1,c.2210A > T) were found in similar proportions in the mRNA as in the genomic DNA. We conclude that the mRNA examination should precede functional tests at protein level. PMID:16341550
Present and future of antisense therapy for splicing modulation in inherited metabolic disease
Belen P�rez; Laura Rodr�guez-Pascau; Luisa Vilageliu; Daniel Grinberg; Magdalena Ugarte; Lourdes R. Desviat
The number of mutations identified deep in introns which activate or create novel splice sites resulting in pathogenic pseudoexon\\u000a inclusion in mRNA continues to grow for inherited metabolic disease (IMD) and other human genetic diseases. A common characteristic\\u000a is that the native splice sites remain intact thus retaining the potential for normal splicing. Antisense oligonucleotides\\u000a (AO) have been shown to
Variation in sequence and organization of splicing regulatory elements in vertebrate genes
Yeo, Gene; Hoon, Shawn; Venkatesh, Byrappa; Burge, Christopher B.
Although core mechanisms and machinery of premRNA splicing are conserved from yeast to human, the details of intron recognition often differ, even between closely related organisms. For example, genes from the pufferfish Fugu rubripes generally contain one or more introns that are not properly spliced in mouse cells. Exploiting available genome sequence data, a battery of sequence analysis techniques was used to reach several conclusions about the organization and evolution of splicing regulatory elements in vertebrate genes. The classical splice site and putative branch site signals are completely conserved across the vertebrates studied (human, mouse, pufferfish, and zebrafish), and exonic splicing enhancers also appear broadly conserved in vertebrates. However, another class of splicing regulatory elements, the intronic splicing enhancers, appears to differ substantially between mammals and fish, with G triples (GGG) very abundant in mammalian introns but comparatively rare in fish. Conversely, short repeats of AC and GT are predicted to function as intronic splicing enhancers in fish but are not enriched in mammalian introns. Consistent with this pattern, exonic splicing enhancer-binding SR proteins are highly conserved across all vertebrates, whereas heterogeneous nuclear ribonucleoproteins, which bind many intronic sequences, vary in domain structure and even presence/absence between mammals and fish. Exploiting differences in intronic sequence composition, a statistical model was developed to predict the splicing phenotype of Fugu introns in mammalian systems and was used to engineer the spliceability of a Fugu intron in human cells by insertion of specific sequences, thereby rescuing splicing in human cells. PMID:15505203
Analysis of pollen-specific alternative splicing in Arabidopsis thaliana via semi-quantitative PCR.
Estrada, April D; Freese, Nowlan H; Blakley, Ivory C; Loraine, Ann E
Alternative splicing enables a single gene to produce multiple mRNA isoforms by varying splice site selection. In animals, alternative splicing of mRNA isoforms between cell types is widespread and supports cellular differentiation. In plants, at least 20% of multi-exon genes are alternatively spliced, but the extent and significance of tissue-specific splicing is less well understood, partly because it is difficult to isolate cells of a single type. Pollen is a useful model system to study tissue-specific splicing in higher plants because pollen grains contain only two cell types and can be collected in large amounts without damaging cells. Previously, we identified pollen-specific splicing patterns by comparing RNA-Seq data from Arabidopsis pollen and leaves. Here, we used semi-quantitative PCR to validate pollen-specific splicing patterns among genes where RNA-Seq data analysis indicated splicing was most different between pollen and leaves. PCR testing confirmed eight of nine alternative splicing patterns, and results from the ninth were inconclusive. In four genes, alternative transcriptional start sites coincided with alternative splicing. This study highlights the value of the low-cost PCR assay as a method of validating RNA-Seq results. PMID:25945312
A new large animal model of CLN5 neuronal ceroid lipofuscinosis in Borderdale sheep is caused by a nucleotide substitution at a consensus splice site (c.571+1G>A) leading to excision of exon 3
Frugier, Tony; Mitchell, Nadia L.; Tammen, Imke; Houweling, Peter J.; Arthur, Donald G.; Kay, Graham W.; van Diggelen, Otto P.; Jolly, Robert D.; Palmer, David N.
Batten disease (neuronal ceroid lipofuscinoses, NCLs) are a group of inherited childhood diseases that result in severe brain atrophy, blindness and seizures, leading to premature death. To date eight different genes have been identified, each associated with a different form. Linkage analysis indicated a CLN5 form in a colony of affected New Zealand Borderdale sheep. Sequencing studies established the disease-causing mutation to be a substitution at a consensus splice site (c.571+G>A), leading to the excision of exon 3 and a truncated putative protein. A molecular diagnostic test has been developed based on the excision of exon 3. Sequence alignments support the gene product being a soluble lysosomal protein. Western blotting of isolated storage bodies indicates the specific storage of subunit c of mitochondrial ATP synthase. This flock is being expanded as a large animal model for mechanistic studies and trial therapies. PMID:17988881
Redirecting splicing with bifunctional oligonucleotides
Perreault, Jean-Pierre
oligonucleotides made of an antisense portion that determines target specificity, and a non- hybridizing tailRedirecting splicing with bifunctional oligonucleotides Jean-Philippe Brosseau1,2 , Jean-Franc� ois, for targeted oligonucleotide silencer of splicing and targeted oligonucleotide enhancer of splicing). The use
Designing oligo libraries taking alternative splicing into account
NASA Astrophysics Data System (ADS)
Shoshan, Avi; Grebinskiy, Vladimir; Magen, Avner; Scolnicov, Ariel; Fink, Eyal; Lehavi, David; Wasserman, Alon
We have designed sequences for DNA microarrays and oligo libraries, taking alternative splicing into account. Alternative splicing is a common phenomenon, occurring in more than 25% of the human genes. In many cases, different splice variants have different functions, are expressed in different tissues or may indicate different stages of disease. When designing sequences for DNA microarrays or oligo libraries, it is very important to take into account the sequence information of all the mRNA transcripts. Therefore, when a gene has more than one transcript (as a result of alternative splicing, alternative promoter sites or alternative poly-adenylation sites), it is very important to take all of them into account in the design. We have used the LEADS transcriptome prediction system to cluster and assemble the human sequences in GenBank and design optimal oligonucleotides for all the human genes with a known mRNA sequence based on the LEADS predictions.
Splice assembly tool and method of splicing
Silva, Frank A. (Basking Ridge, NJ)
A splice assembly tool for assembling component parts of an electrical conductor while producing a splice connection between electrical cables therewith, comprises a first structural member adaptable for supporting force applying means thereon, said force applying means enabling a rotary force applied manually thereto to be converted to a longitudinal force for subsequent application against a first component part of said electrical connection, a second structural member adaptable for engaging a second component part in a manner to assist said first structural member in assembling the component parts relative to one another and transmission means for conveying said longitudinal force between said first and said second structural members, said first and said second structural members being coupled to one another by said transmission means, wherein at least one of said component parts comprises a tubular elastomeric sleeve and said force applying means provides a relatively high mechanical advantage when said rotary force is applied thereto so as to facilitate assembly of said at least one tubular elastomeric sleeve about said other component part in an interference fit manner.
FOA Lecture 6: Fiber Optic Splices
NSDL National Science Digital Library
This is the sixth lecture in the FOA series on fiber optics, covering splices. In this lecture the presenter discusses the uses of splices, types of splices such as fusion splices and mechanical splices, and the processes used for making each type of splice. The video also discusses cleaving, which is the key to getting good splices. Running time for the lecture is 9:03. Flash is required to view the video.
Distinct regulatory programs establish widespread sex-specific alternative splicing in Drosophila melanogaster
Hartmann, Britta; Castelo, Robert; Mi�ana, Bel�n; Peden, Erin; Blanchette, Marco; Rio, Donald C.; Singh, Ravinder; Valc�rcel, Juan
In Drosophila melanogaster, female-specific expression of Sex-lethal (SXL) and Transformer (TRA) proteins controls sex-specific alternative splicing and/or translation of a handful of regulatory genes responsible for sexual differentiation and behavior. Recent findings in 2009 by Telonis-Scott et al. document widespread sex-biased alternative splicing in fruitflies, including instances of tissue-restricted sex-specific splicing. Here we report results arguing that some of these novel sex-specific splicing events are regulated by mechanisms distinct from those established by female-specific expression of SXL and TRA. Bioinformatic analysis of SXL/TRA binding sites, experimental analysis of sex-specific splicing in S2 and Kc cells lines and of the effects of SXL knockdown in Kc cells indicate that SXL-dependent and SXL-independent regulatory mechanisms coexist within the same cell. Additional determinants of sex-specific splicing can be provided by sex-specific differences in the expression of RNA binding proteins, including Hrp40/Squid. We report that sex-specific alternative splicing of the gene hrp40/squid leads to sex-specific differences in the levels of this hnRNP protein. The significant overlap between sex-regulated alternative splicing changes and those induced by knockdown of hrp40/squid and the presence of related sequence motifs enriched near subsets of Hrp40/Squid-regulated and sex-regulated splice sites indicate that this protein contributes to sex-specific splicing regulation. A significant fraction of sex-specific splicing differences are absent in germline-less tudor mutant flies. Intriguingly, these include alternative splicing events that are differentially spliced in tissues distant from the germline. Collectively, our results reveal that distinct genetic programs control widespread sex-specific splicing in Drosophila melanogaster. PMID:21233220
Modeling the active sites of non-heme diiron metalloproteins with sterically hindered carboxylates and syn N-Donor ligands
Friedle, Simone, 1976-
Chapter 1. Different Synthetic Approaches to Modeling the Active Sites of Carboxylate-Bridged Non-Heme Diiron Enzymes Carboxylate-bridged non-heme diiron enzymes activate dioxygen to perform a variety of biological functions. ...
Oxidation of substrates tethered to N-donor ligands for modeling non-heme diiron enzyme active sites
Carson, Emily Carrig, 1978-
Chapter 1. Modeling Carboxylate-Rich Diiron Sites of Dioxygen-Dependent Non-Heme Enzymes Carboxylate-bridged diiron centers are employed in a variety of biological systems to activate dioxygen for substrate oxidation, and ...
Splicing-Related Features of Introns Serve to Propel Evolution
Luo, Yuping; Li, Chun; Gong, Xi; Wang, Yanlu; Zhang, Kunshan; Cui, Yaru; Sun, Yi Eve; Li, Siguang
The role of spliceosomal intronic structures played in evolution has only begun to be elucidated. Comparative genomic analyses of fungal snoRNA sequences, which are often contained within introns and/or exons, revealed that about one-third of snoRNA-associated introns in three major snoRNA gene clusters manifested polymorphisms, likely resulting from intron loss and gain events during fungi evolution. Genomic deletions can clearly be observed as one mechanism underlying intron and exon loss, as well as generation of complex introns where several introns lie in juxtaposition without intercalating exons. Strikingly, by tracking conserved snoRNAs in introns, we found that some introns had moved from one position to another by excision from donor sites and insertion into target sties elsewhere in the genome without needing transposon structures. This study revealed the origin of many newly gained introns. Moreover, our analyses suggested that intron-containing sequences were more prone to sustainable structural changes than DNA sequences without introns due to intron's ability to jump within the genome via unknown mechanisms. We propose that splicing-related structural features of introns serve as an additional motor to propel evolution. PMID:23516505
Multiple factors influence the normal and UV-inducible alternative splicing of PIG3.
Nicholls, Chris D; Beattie, Tara L
In addition to normal alternative splicing events (those that take place in untreated cells), there are also those that are inducible in response to environmental stimuli. We previously reported that the alternative splicing of p53-inducible gene 3 (PIG3) exon 4 is modulated in response to UV radiation. Here, we investigate the mechanisms mediating the alternative splicing of this exon. Through the use of various minigene constructs our results reveal that numerous factors influence the normal alternative splicing of PIG3 exon 4, and that these ultimately affect its UV-inducible alternative splicing. Included among these are sequence elements located within exon 4 itself as well as adjacent sequences required for intron definition (the pyrimidine tract, 3'- and 5'-splice sites). Within exon 4, we identified a novel hnRNP A1-dependent exonic splicing silencer (ESS) whose mutation inhibited the alternative splicing of a PIG3 minigene. Although previously implicated in the UV-inducible alternative splicing of other transcripts, RNAi-mediated silencing of hnRNP A1/A2 or hSlu7 did not prevent the UV-enhancement of exon 4 skipping. Overall, our results suggest that numerous factors contribute to the normal alternative splicing of PIG3 exon 4 and that UV-inducible increases in this process require that the splicing of this exon be maintained in a sufficiently weakened state under normal conditions. PMID:18801469
Design principles for bifunctional targeted oligonucleotide enhancers of splicing
Owen, Nicholas; Zhou, Haiyan; Malygin, Alexey A.; Sangha, Jason; Smith, Lindsay D.; Muntoni, Francesco; Eperon, Ian C.
Controlling the patterns of splicing of specific genes is an important goal in the development of new therapies. We have shown that the splicing of a refractory exon, SMN2 exon 7, could be increased in fibroblasts derived from patients with spinal muscular atrophy by using bifunctional targeted oligonucleotide enhancers of splicing (TOES) oligonucleotides that anneal to the exon and contain a �tail� of enhancer sequences that recruit activating proteins. We show here that there are striking agreements between the effects of oligonucleotides on splicing in vitro and on both splicing and SMN2 protein expression in patient-derived fibroblasts, indicating that the effects on splicing are the major determinant of success. Increased exon inclusion depends on the number, sequence and chemistry of the motifs that bind the activator protein SRSF1, but it is not improved by increasing the strength of annealing to the target site. The optimal oligonucleotide increases protein levels in transfected fibroblasts by a mean value of 2.6-fold (maximum 4.6-fold), and after two rounds of transfection the effect lasted for a month. Oligonucleotides targeted to the upstream exon (exon 6 in SMN) are also effective. We conclude that TOES oligonucleotides are highly effective reagents for restoring the splicing of refractory exons and can act across long introns. PMID:21602265
Genome-wide survey of Alternative Splicing in Sorghum Bicolor.
Panahi, Bahman; Abbaszadeh, Bahram; Taghizadeghan, Mehdi; Ebrahimie, Esmaeil
Sorghum bicolor is a member of grass family which is an attractive model plant for genome study due to interesting genome features like low genome size. In this research, we performed comprehensive investigation of Alternative Splicing and ontology aspects of genes those have undergone these events in sorghum bicolor. We used homology based alignments between gene rich transcripts, represented by tentative consensus (TC) transcript sequences, and genomic scaffolds to deduce the structure of genes and identify alternatively spliced transcripts in sorghum. Using homology mapping of assembled expressed sequence tags with genomics data, we identified 2,137 Alternative Splicing events in S. bicolor. Our study showed that complex events and intron retention are the main types of Alternative Splicing events in S. bicolor and highlights the prevalence of splicing site recognition for definition of introns in this plant. Annotations of the alternatively spliced genes revealed that they represent diverse biological process and molecular functions, suggesting a fundamental role for Alternative Splicing in affecting the development and physiology of S. bicolor. PMID:25049459
Genome-Wide Analysis of Heat-Sensitive Alternative Splicing in Physcomitrella patens.
Chang, Chiung-Yun; Lin, Wen-Dar; Tu, Shih-Long
Plant growth and development are constantly influenced by temperature fluctuations. To respond to temperature changes, different levels of gene regulation are modulated in the cell. Alternative splicing (AS) is a widespread mechanism increasing transcriptome complexity and proteome diversity. Although genome-wide studies have revealed complex AS patterns in plants, whether AS impacts the stress defense of plants is not known. We used heat shock (HS) treatments at nondamaging temperature and messenger RNA sequencing to obtain HS transcriptomes in the moss Physcomitrella patens. Data analysis identified a significant number of novel AS events in the moss protonema. Nearly 50% of genes are alternatively spliced. Intron retention (IR) is markedly repressed under elevated temperature but alternative donor/acceptor site and exon skipping are mainly induced, indicating differential regulation of AS in response to heat stress. Transcripts undergoing heat-sensitive IR are mostly involved in specific functions, which suggests that plants regulate AS with transcript specificity under elevated temperature. An exonic GAG-repeat motif in these IR regions may function as a regulatory cis-element in heat-mediated AS regulation. A conserved AS pattern for HS transcription factors in P. patens and Arabidopsis (Arabidopsis thaliana) reveals that heat regulation for AS evolved early during land colonization of green plants. Our results support that AS of specific genes, including key HS regulators, is fine-tuned under elevated temperature to modulate gene regulation and reorganize metabolic processes. PMID:24777346
RNA splicing mutation in an aberrantly rearranged immunoglobulin lambda I gene.
Hozumi, N; Wu, G E; Murialdo, H; Roberts, L; Vetter, D; Fife, W L; Whiteley, M; Sadowski, P
The mouse cell line MOPC 315 is an IgA (lambda II)-producing myeloma. We have studied a derivative of MOPC 315 that secretes normal lambda II chains but no heavy chain. This derivative, MOPC 315-26, was found to contain a rearranged lambda I gene in addition to a rearranged lambda II gene. The rearranged lambda I gene was cloned into bacteriophage lambda DNA and its structure was studied. The lambda I gene was found to have arisen by an aberrant recombination event that resulted in a single base insertion at the site of V-J region joining. In addition, the gene contained numerous point mutations in the vicinity of the junction of the V and J regions. Two point mutations occurred in the donor splice sequence normally used for the removal of the intron between the J and C regions, suggesting that the RNA synthesized from the aberrantly rearranged lambda I gene would be unable to undergo proper RNA splicing. Images PMID:6171827
Biogeochemical Modeling of In Situ U(VI) Reduction and Immobilization with Emulsified Vegetable Oil as the Electron Donor at a Field Site in Oak Ridge, Tennessee
NASA Astrophysics Data System (ADS)
Tang, G.; Parker, J.; Wu, W.; Schadt, C. W.; Watson, D. B.; Brooks, S. C.; Orifrc Team
A comprehensive biogeochemical model was developed to quantitatively describe the coupled hydrologic, geochemical and microbiological processes that occurred following injection of emulsified vegetable oil (EVO) as the electron donor to immobilize U(VI) at the Oak Ridge Integrated Field Research Challenge site (ORIFRC) in Tennessee. The model couples the degradation of EVO, production and oxidation of long-chain fatty acids (LCFA), glycerol, hydrogen and acetate, reduction of nitrate, manganese, ferrous iron, sulfate and uranium, and methanoganesis with growth of multiple microbial groups. The model describes the evolution of geochemistry and microbial populations not only in the aqueous phase as typically observed, but also in the mineral phase and therefore enables us to evaluate the applicability of rates from the literature for field scale assessment, estimate the retention and degradation rates of EVO and LCFA, and assess the influence of the coupled processes on fate and transport of U(VI). Our results suggested that syntrophic bacteria or metal reducers might catalyze LCFA oxidation in the downstream locations when sulfate was consumed, and competition between methanogens and others for electron donors and slow growth of methanogen might contribute to the sustained reducing condition. Among the large amount of hydrologic, geochemical and microbiological parameter values, the initial biomass, and the interactions (e.g., inhibition) of the microbial functional groups, and the rate and extent of Mn and Fe oxide reduction appear as the major sources of uncertainty. Our model provides a platform to conduct numerical experiments to study these interactions, and could be useful for further iterative experimental and modeling investigations into the bioreductive immobiliztion of radionuclide and metal contaminants in the subsurface.
Novel splice variants of the bovine PCK1 gene.
Zhang, Z B; Zhang, W; Li, R L; Li, J B; Zhong, J F; Zhao, Z S; Huang, J M
Phosphoenolpyruvate carboxykinase 1 (PCK1), also named PEPCK-C, is a multiple-function gene that is involved in gluconeogenesis, glyceroneogenesis, reproduction, female fertility, and development of obesity and diabetes. How its many functions are regulated was largely unknown. Therefore, we investigated mRNA expression and possible splice variants of PCK1 by screening cDNA in nine tissues from Holstein bulls and cows. PCK1 mRNA was highly expressed in the liver, kidney, ovary and testis; expression levels were low in the heart, spleen, and lung tissues. Expression of this gene was not detected in skeletal muscle. This led to the discovery of five novel bovine splice variants, named PCK1-AS1-PCK1-AS5. In PCK1-AS1, 51 nucleotides in the interior of exon 2 were spliced out. In PCK1-AS2, exons 2 and 3 were altered by the alternative 3' and 5' splice sites, respectively. PCK1-AS3 was truncated from the 3' end of exon 2 to the 5' end of exon 4. In PCK1-AS4, exon 5 was completely spliced out. In PCK1-AS5, exons 5 and 6 and the 5' end of exon 7 were spliced out. These splice variants (PCK1-AS1-PCK1-AS5) potentially encoded shorter proteins (605, 546, 373, 246 and 274 amino acids, respectively), when compared to the complete protein (622 amino acids). Considering the functional domains of the PCK1 protein, it is likely that these splice variants considerably affect the function of this protein; alternative splicing could be one of the mechanisms by which the diverse functions of PCK1 are regulated. PMID:24089092
Genome-wide detection of alternative splicing in expressed sequences of human genes
Barmak Modrek; Alissa Resch; Catherine Grasso; Christopher Lee
We have identified 6201 alternative splice relation- ships in human genes, through a genome-wide analysis of expressed sequence tags (ESTs). Starting with ?2.1 million human mRNA and EST sequences, we mapped expressed sequences onto the draft human genome sequence and only accepted splices that obeyed the standard splice site consensus. A large fraction (47%) of these were observed multiple times,
TRAP150 activates splicing in composite terminal exons
Lee, Kuo-Ming; Tarn, Woan-Yuh
The spliceosomal factor TRAP150 is essential for pre-mRNA splicing in vivo and, when overexpressed, it enhances splicing efficiency. In this study, we found that TRAP150 interacted with the cleavage and polyadenylation specificity factor (CPSF) and co-fractionated with CPSF and RNA polymerase II. Moreover, TRAP150 preferentially associated with the U1 small ribonucleoprotein (snRNP). However, our data do not support a role for TRAP150 in alternative 5? splice site or exon selection or in alternative polyadenylation. Because U1 snRNP participates in premature cleavage and polyadenylation (PCPA), we tested whether TRAP150 is a cofactor in the control of PCPA. Although TRAP150 depletion had no significant effect on PCPA, overexpression of TRAP150 forced activation of a cryptic 3? splice site, yielding spliced PCPA transcripts. Mechanistic studies showed that TRAP150-activated splicing occurred in composite but not authentic terminal exons, and such an activity was enhanced by debilitation of U1 snRNP or interference with transcription elongation or termination. Together, these results indicate that TRAP150 provides an additional layer of PCPA regulation, through which it may increase the diversity of abortive RNA transcripts under conditions of compromised gene expression. PMID:25326322
Splicing Wires Permanently With Explosives
NASA Technical Reports Server (NTRS)
Bement, Laurence J.; Kushnick, Anne C.
Explosive joining process developed to splice wires by enclosing and metallurgically bonding wires within copper sheets. Joints exhibit many desirable characteristics, 100-percent conductivity and strength, no heat-induced annealing, no susceptibility to corrosion in contacts between dissimilar metals, and stability at high temperature. Used to join wires to terminals, as well as to splice wires. Applicable to telecommunications industry, in which millions of small wires spliced annually.
Modification of MyD88 mRNA splicing and inhibition of IL-1beta signaling in cell culture and in mice with a 2'-O-methoxyethyl-modified oligonucleotide.
Vickers, Timothy A; Zhang, Hong; Graham, Mark J; Lemonidis, Kristina M; Zhao, Chenguang; Dean, Nicholas M
A number of proinflammatory cytokines, including IL-1beta, signal through the adaptor protein MyD88. This signaling leads to phosphorylation of IL-1R-associated kinase-1 (IRAK-1) and, ultimately, activation of the NF-kappaB transcription factor. A splice variant of MyD88 (MyD88(S)), which lacks the ability to couple IRAK-1 to NF-kappaB, has been described. A chemically modified antisense oligonucleotide (ASO) that alters the splicing ratio of MyD88 to MyD88(S) in both cell culture and in animals has been identified. The ASO (ISIS 337846) binds to exon II donor sites in the MyD88 pre-mRNA. By manipulating levels of MyD88 splicing, proinflammatory signaling through the IL-1R has been shown to be diminished, both in cell culture and in mouse liver. To our knowledge, this represents the first example of modulation of RNA splicing of an endogenous gene target in animals after systemic ASO dosing and suggests that this mechanism may be useful as a novel modulator of inflammatory stimuli. PMID:16517734
Management of Young Blood Donors
Newman, Bruce H.
Summary The emphasis on high-school blood drives and acceptance of 16-year-old blood donors led to more research on physiologic and psychological ways to decrease vasovagal reaction rates in young blood donors and to increase donor retention. Research on how to accomplish this has been advantageous for the blood collection industry and blood donors. This review discussed the current situation and what can be done psychologically, physiologically, and via process improvements to decrease vasovagal reaction rates and increase donor retention. The donation process can be significantly improved. Future interventions may include more dietary salt, a shorter muscle tension program to make it more feasible, recommendations for post-donation muscle tension / squatting / laying down for lightheadedness, more donor education by the staff at the collection site, more staff attention to donors with fear or higher risk for a vasovagal reaction (e.g. estimated blood volume near 3.5 l, first-time donor), and a more focused donation process to ensure a pleasant and safer procedure. PMID:25254024
Management of young blood donors.
Newman, Bruce H
The emphasis on high-school blood drives and acceptance of 16-year-old blood donors led to more research on physiologic and psychological ways to decrease vasovagal reaction rates in young blood donors and to increase donor retention. Research on how to accomplish this has been advantageous for the blood collection industry and blood donors. This review discussed the current situation and what can be done psychologically, physiologically, and via process improvements to decrease vasovagal reaction rates and increase donor retention. The donation process can be significantly improved. Future interventions may include more dietary salt, a shorter muscle tension program to make it more feasible, recommendations for post-donation muscle tension / squatting / laying down for lightheadedness, more donor education by the staff at the collection site, more staff attention to donors with fear or higher risk for a vasovagal reaction (e.g. estimated blood volume near 3.5 l, first-time donor), and a more focused donation process to ensure a pleasant and safer procedure. PMID:25254024
Identification of a homozygous splice site mutation in the dynein axonemal light chain 4 gene on 22q13.1 in a large consanguineous family from Pakistan with congenital mirror movement disorder.
Ahmed, Iltaf; Mittal, Kirti; Sheikh, Taimoor I; Vasli, Nasim; Rafiq, Muhammad Arshad; Mikhailov, Anna; Ohadi, Mehrnaz; Mahmood, Huda; Rouleau, Guy A; Bhatti, Attya; Ayub, Muhammad; Srour, Myriam; John, Peter; Vincent, John B
Mirror movements (MRMV) are involuntary movements on one side of the body that mirror voluntary movements on the opposite side. Congenital mirror movement disorder is a rare, typically autosomal-dominant disorder, although it has been suspected that some sporadic cases may be due to recessive inheritance. Using a linkage analysis and a candidate gene approach, two genes have been implicated in congenital MRMV disorder to date: DCC on 18q21.2 (MRMV1), which encodes a netrin receptor, and RAD51 on 15q15.1 (MRMV2), which is involved in the maintenance of genomic integrity. Here, we describe a large consanguineous Pakistani family with 11 cases of congenital MRMV disorder reported across five generations, with autosomal recessive inheritance likely. Sanger sequencing of DCC and RAD51 did not identify a mutation. We then employed microarray genotyping and autozygosity mapping to identify a shared region of homozygosity-by-descent among the affected individuals. We identified a large autozygous region of ~3.3 Mb on chromosome 22q13.1 (Chr22:36605976-39904648). We used Sanger sequencing to exclude several candidate genes within this region, including DMC1 and NPTXR. Whole exome sequencing was employed, and identified a splice site mutation in the dynein axonemal light chain 4 gene, DNAL4. This splice site change leads to skipping of exon 3, and omission of 28 amino acids from DNAL4 protein. Linkage analysis using Simwalk2 gives a maximum Lod score of 6.197 at this locus. Whether or how DNAL4 function may relate to the function of DCC or RAD51 is not known. Also, there is no suggestion of primary ciliary dyskinesis, situs inversus, or defective sperm in affected family members, which might be anticipated given a putative role for DNAL4 in axonemal-based dynein complexes. We suggest that DNAL4 plays a role in the cytoplasmic dynein complex for netrin-1-directed retrograde transport, and in commissural neurons of the corpus callosum in particular. This, in turn, could lead to faulty cross-brain wiring, resulting in MRMV. PMID:25098561
Basal Splicing Factors Regulate the Stability of Mature mRNAs in Trypanosomes*
Gupta, Sachin Kumar; Carmi, Shai; Ben-Asher, Hiba Waldman; Tkacz, Itai Dov; Naboishchikov, Ilana; Michaeli, Shulamit
Gene expression in trypanosomes is mainly regulated post-transcriptionally. Genes are transcribed as polycistronic mRNAs that are dissected by the concerted action of trans-splicing and polyadenylation. In trans-splicing, a common exon, the spliced leader, is added to all mRNAs from a small RNA. In this study, we examined by microarray analysis the transcriptome following RNAi silencing of the basal splicing factors U2AF65, SF1, and U2AF35. The transcriptome data revealed correlations between the affected genes and their splicing and polyadenylation signaling properties, suggesting that differential binding of these factors to pre-mRNA regulates trans-splicing and hence expression of specific genes. Surprisingly, all these factors were shown to affect not only splicing but also mRNA stability. Affinity purification of SF1 and U2AF35 complexes supported their role in mRNA stability. U2AF35 but not SF1 was shown to bind to ribosomes. To examine the role of splicing factors in mRNA stability, mutations were introduced into the polypyrimidine tract located in the 3? UTR of a mini-gene, and the results demonstrate that U2AF65 binds to such a site and controls the mRNA stability. We propose that transcripts carrying splicing signals in their 3? UTR bind the splicing factors and control their stability. PMID:23283975
Accurate splicing of HDAC6 pre-mRNA requires SON.
Battini, Vishnu Priya; Bubulya, Athanasios; Bubulya, Paula A
Pre-mRNA splicing requires proper splice site selection mediated by many factors including snRNPs and serine-arginine rich (SR) splicing factors. Our lab previously reported that the SR-like protein SON maintains organization of pre-mRNA splicing factors in nuclear speckles as well as splicing of many human transcripts including mRNAs coding for the chromatin-modifying enzymes HDAC6, ADA and SETD8. However, the mechanism by which SON maintains accurate splicing is unknown. To build tools for understanding SON-dependent pre-mRNA splicing, we constructed a minigene reporter plasmid driving expression of the genomic sequence spanning exons 26 through 29 of HDAC6. Following SON depletion, we observed altered splicing of HDAC6 reporter transcripts that showed exclusion of exons 27 and 28, reflecting the splicing patterns of endogenous HDAC6 mRNA. Importantly, loss of HDAC6 biological function was also observed, as indicated by truncated HDAC6 protein and corresponding absence of aggresome assembly activities of HDAC6 binding-of-ubiquitin zinc finger (BUZ) domain. We therefore propose that SON-mediated splicing regulation of HDAC6 is essential for supporting protein degradation pathways that prevent human disease. PMID:25782155
Molecular Outcome, Prediction, and Clinical Consequences of Splice Variants in COL1A1, Which Encodes the pro?1(I) Chains of Type I Procollagen.
Schleit, Jennifer; Bailey, Samuel S; Tran, Thao; Chen, Diana; Stowers, Susan; Schwarze, Ulrike; Byers, Peter H
Approximately 10%-20% of germline pathogenic variants alter mRNA splicing, with phenotypes often dependent on the stability of the mRNA produced by the mutant allele. To better understand the relationships between genotype, mRNA splicing, and phenotype, we examined clinical and molecular data from 243 probands with osteogenesis imperfecta (OI) representing 145 unique splicing variants within the type I procollagen gene, COL1A1. All individuals with IVSX-1G>A mutations had OI type I because the substitution shifted the splice acceptor site 1 nt downstream and destabilized the mRNA. OI phenotypes were not consistent for any other splice variant identified. We sequenced all cDNA species from cultured dermal fibroblasts from 40 individuals to identify splice outcome and compared those results to splice predictions from Human Splice Finder (HSF), Spliceport (SP), and Automatic Splice Site and Exon Definition Analyses (ASSEDA). Software-based splice predictions were correct in 42%, 55%, and 74% instances for HSF, SP, and ASSEDA, respectively. As molecular diagnostics move increasingly to DNA sequence analysis, the need to understand the effects of splice site variants will increase. These data demonstrate that caution must be exercised when using splice prediction software to predict splice outcome. PMID:25963598
nagnag: Identification and quantification of NAGNAG alternative splicing using RNA-Seq data.
Yan, Xiaoyan; Sablok, Gaurav; Feng, Gang; Ma, Jiaxin; Zhao, Hongwei; Sun, Xiaoyong
Regulation of proteome diversity by alternative splicing has been widely demonstrated in plants and animals. NAGNAG splicing, which was recently defined as a tissue specific event, results in the production of two distinct isoforms that are distinguished by three nucleotides (NAG) as a consequence of the intron proximal or distal to the splice site. Since the NAGNAG mechanism is not well characterized, tools for the identification and quantification of NAGNAG splicing events remain under-developed. Here we report nagnag, an R-based NAGNAG splicing detection tool, which accurately identifies and quantifies NAGNAG splicing events using RNA-Seq. Overall, nagnag produces user-friendly visualization reports and highlights differences between the DNA/RNA/protein across the identified isoforms of the reported gene. The package is available on https://sourceforge.net/projects/nagnag/files/; or http://genome.sdau.edu.cn/research/software/nagnag.html. PMID:26028313
Committee of Donor Agencies for Small Enterprise Development
NSDL National Science Digital Library
Established in 1979, the Committee of Donor Agencies promotes the development of small enterprise in developing countries. This site offers numerous working and research papers about the Committee of Donor Agencies and its members. Showcased on the site are the Committee's Donor Business Development Services Case Studies. The Case studies are browseable by several categories including Region, Country, Theme, and Member Agency. Also provided are the Donor Committee Guidelines and links to member agencies's sites.
Discovery and characterization of secretory IgD in rainbow trout: secretory IgD is produced through a novel splicing mechanism
Ramirez-Gomez, F.; Greene, W.; Rego, K.; Hansen, J.D.; Costa, G.; Kataria, P.; Bromage, E.S.
The gene encoding IgH ? has been found in all species of teleosts studied to date. However, catfish (Ictalurus punctatus) is the only species of fish in which a secretory form of IgD has been characterized, and it occurs through the use of a dedicated ?-secretory exon, which is absent from all other species examined. Our studies have revealed that rainbow trout (Oncorhynchus mykiss) use a novel strategy for the generation of secreted IgD. The trout secretory ? transcript is produced via a run-on event in which the splice donor site at the end of the last constant domain exon (D7) is ignored and transcription continues until a stop codon is reached 33 nt downstream of the splice site, resulting in the production of an in-frame, 11-aa secretory tail at the end of the D7 domain. In silico analysis of several published IgD genes suggested that this unique splicing mechanism may also be used in other species of fish, reptiles, and amphibians. Alternative splicing of the secretory ? transcript resulted in two ?-H chains, which incorporated C?1 and variable domains. Secreted IgD was found in two heavily glycosylated isoforms, which are assembled as monomeric polypeptides associated with L chains. Secretory ? mRNA and IgD+ plasma cells were detected in all immune tissues at a lower frequency than secretory IgM. Our data demonstrate that secretory IgD is more prevalent and widespread across taxa than previously thought, and thus illustrate the potential that IgD may have a conserved role in immunity.
Classifying MLH1 and MSH2 variants using bioinformatic prediction, splicing assays, segregation and tumor characteristics
Arnold, Sven; Buchanan, Daniel D.; Barker, Melissa; Jaskowski, Lesley; Walsh, Michael D.; Birney, Genevieve; Woods, Michael O.; Hopper, John L.; Jenkins, Mark A.; Brown, Melissa A.; Tavtigian, Sean V.; Goldgar, David E.; Young, Joanne P.; Spurdle, Amanda B.
Reliable methods for predicting functional consequences of variants in disease genes would be beneficial in the clinical setting. This study was undertaken to predict, and confirm in vitro, splicing aberrations associated with mismatch repair (MMR) variants identified in familial colon cancer patients. Six programs were used to predict the effect of 13 MLH1 and 6 MSH2 gene variants on pre-mRNA splicing. mRNA from cycloheximide-treated lymphoblastoid cell lines of variant carriers was screened for splicing aberrations. Tumors of variant carriers were tested for microsatellite instability and MMR protein expression. Variant segregation in families was assessed using Bayes factor causality analysis. Amino acid alterations were examined for evolutionary conservation and physicochemical properties. Splicing aberrations were detected for ten variants, including a frameshift as a minor cDNA product, and altered ratio of known alternate splice products. Loss of splice sites was well predicted by splice site prediction programs SpliceSiteFinder (90%) and NNSPLICE (90%), but consequence of splice site loss was less accurately predicted. No aberrations correlated with ESE predictions for the nine exonic variants studied. Seven of eight missense variants had normal splicing (88%), but only one was a substitution considered neutral from evolutionary/physicochemical analysis. Combined with information from tumor and segregation analysis, and literature review, 16/19 variants were considered clinically relevant. Bioinformatic tools for prediction of splicing aberrations need improvement before use without supporting studies to assess variant pathogenicity. Classification of mismatch repair gene variants is assisted by a comprehensive approach which includes in vitro, tumor pathology, clinical, and evolutionary conservation data. PMID:19267393
Processing of fish lg heavy chain transcripts diverse splicing patterns and unusual nonsense mediated decay
Technology Transfer Automated Retrieval System (TEKTRAN)
Alternate pathways of RNA processing play an important role in the expression of the secreted (S) and membrane (Mb) forms of immunoglobulin (Ig) heavy (H) chain isotypes in all vertebrates. Interestingly, while the differential splicing mechanism and the splice sites that generate the two forms of I...
Evidence for Widespread Association of Mammalian Splicing and Conserved Long-Range RNA Structures
Pervouchine, Dmitri D.
of Physics and Technology, Moscow, 141700, Russia 4Institute of Molecular Biology and Genetics NAS of Ukraine splice sites. As an example, we validated the RNA structure in the human SF1 gene using mini-genes in HEK exons 9 and 10 of this gene altered the splicing pattern, while the compensatory mutations that re
Cleavage of intron from the standard or non-standard position of the precursor tRNA by the splicing endonuclease of Aeropyrum pernix, a hyper-thermophilic Crenarchaeon, involves a novel RNA recognition site in the Crenarchaea specific loop
Hirata, Akira; Kitajima, Tsubasa; Hori, Hiroyuki
In Crenarchaea, several tRNA genes are predicted to express precursor-tRNAs (pre-tRNAs) with canonical or non-canonical introns at various positions. We initially focused on the tRNAThr species of hyperthermophilic crenarchaeon, Aeropyrum pernix (APE) and found that in the living APE cells three tRNAThr species were transcribed and subsequently matured to functional tRNAs. During maturation, introns in two of them were cleaved from standard and non-standard positions. Biochemical studies revealed that the APE splicing endonuclease (APE-EndA) removed both types of introns, including the non-canonical introns, without any nucleotide modification. To clarify the underlying reasons for broad substrate specificity of APE-EndA, we determined the crystal structure of wild-type APE-EndA and subsequently compared its structure with that of Archaeaoglobus fulgidus (AFU)-EndA, which has narrow substrate specificity. Remarkably, structural comparison revealed that APE-EndA possesses a Crenarchaea specific loop (CSL). Introduction of CSL into AFU-EndA enhanced its intron-cleaving activity irrespective of the position or motif of the intron. Thus, our biochemical and crystallographic analyses of the chimera-EndA demonstrated that the CSL is responsible for the broad substrate specificity of APE-EndA. Furthermore, mutagenesis studies revealed that Lys44 in CSL functions as the RNA recognition site. PMID:21846775
Correspondence Alternative Splicing at NAGNAG
Will, Sebastian
Correspondence Alternative Splicing at NAGNAG Acceptors: Simply Noise or Noise and More? Michael splicing at NAGNAGs mainly results in the insertion/deletion of one amino acid. While such subtle events noise tolerated by cells? Zavolan and colleagues [3,4] suggest that these variations are the result
A Genome-Wide Aberrant RNA Splicing in Patients with Acute Myeloid Leukemia Identifies Novel Potential Disease Markers and Therapeutic Targets
Adamia, Sophia; Haibe-Kains, Benjamin; Pilarski, Patrick M.; Bar-Natan, Michal; Pevzner, Samuel; Avet-Loiseau, Herve; Lode, Laurence; Verselis, Sigitas; Fox, Edward A.; Burke, John; Galinsky, Ilene; Dagogo-Jack, Ibiayi; Wadleigh, Martha; Steensma, David P.; Motyckova, Gabriela; Deangelo, Daniel J.; Quackenbush, John; Stone, Richard; Griffin, James D.
Purpose Despite new treatments, acute myeloid leukemia (AML) remains an incurable disease. More effective drug design requires an expanded view of the molecular complexity that underlies AML. Alternative splicing of RNA is used by normal cells to generate protein diversity. Growing evidence indicates that aberrant splicing of genes plays a key role in cancer. We investigated genome-wide splicing abnormalities in AML and based on these abnormalities, we aimed to identify novel potential biomarkers and therapeutic targets. Experimental Design We used genome-wide alternative splicing screening to investigate alternative splicing abnormalities in two independent AML patient cohorts [Dana-Farber Cancer Institute (DFCI) (Boston, MA) and University Hospital de Nantes (UHN) (Nantes, France)] and normal donors. Selected splicing events were confirmed through cloning and sequencing analysis, and than validated in 193 patients with AML. Results Our results show that approximately 29% of expressed genes genome-wide were differentially and recurrently spliced in patients with AML compared with normal donors bone marrow CD34+ cells. Results were reproducible in two independent AML cohorts. In both cohorts, annotation analyses indicated similar proportions of differentially spliced genes encoding several oncogenes, tumor suppressor proteins, splicing factors, and heterogeneous-nuclear-ribonucleoproteins, proteins involved in apoptosis, cell proliferation, and spliceosome assembly. Our findings are consistent with reports for other malignances and indicate that AML-specific aberrations in splicing mechanisms are a hallmark of AML pathogenesis. Conclusions Overall, our results suggest that aberrant splicing is a common characteristic for AML. Our findings also suggest that splice variant transcripts that are the result of splicing aberrations create novel disease markers and provide potential targets for small molecules or antibody therapeutics for this disease. PMID:24284058
Splice: an analytical network analysis software
Thomas G. Engel
Splice is a symbolic circuit analysis program that operates under the Windows operating system. At the present time, Splice is the only commercially available software that will perform this type of analysis. A description of the features found in Splice 1.0 and examples to illustrate them is presented. Splice works with The Design Center circuit simulator available from the MicroSim
Functional consequences of developmentally regulated alternative splicing
Auinash Kalsotra; Thomas A. Cooper
Genome-wide analyses of metazoan transcriptomes have revealed an unexpected level of mRNA diversity that is generated by alternative splicing. Recently, regulatory networks have been identified through which splicing promotes dynamic remodelling of the transcriptome to promote physiological changes, which involve robust and coordinated alternative splicing transitions. The regulation of splicing in yeast, worms, flies and vertebrates affects a variety of
Unmasking alternative splicing inside protein-coding exons defines exitrons and their role in proteome plasticity
Marquez, Yamile; H�pfler, Markus; Ayatollahi, Zahra; Barta, Andrea; Kalyna, Maria
Alternative splicing (AS) diversifies transcriptomes and proteomes and is widely recognized as a key mechanism for regulating gene expression. Previously, in an analysis of intron retention events in Arabidopsis, we found unusual AS events inside annotated protein-coding exons. Here, we also identify such AS events in human and use these two sets to analyse their features, regulation, functional impact, and evolutionary origin. As these events involve introns with features of both introns and protein-coding exons, we name them exitrons (exonic introns). Though exitrons were detected as a subset of retained introns, they are clearly distinguishable, and their splicing results in transcripts with different fates. About half of the 1002 Arabidopsis and 923 human exitrons have sizes of multiples of 3 nucleotides (nt). Splicing of these exitrons results in internally deleted proteins and affects protein domains, disordered regions, and various post-translational modification sites, thus broadly impacting protein function. Exitron splicing is regulated across tissues, in response to stress and in carcinogenesis. Intriguingly, annotated intronless genes can be also alternatively spliced via exitron usage. We demonstrate that at least some exitrons originate from ancestral coding exons. Based on our findings, we propose a �splicing memory� hypothesis whereby upon intron loss imprints of former exon borders defined by vestigial splicing regulatory elements could drive the evolution of exitron splicing. Altogether, our studies show that exitron splicing is a conserved strategy for increasing proteome plasticity in plants and animals, complementing the repertoire of AS events. PMID:25934563
Unmasking alternative splicing inside protein-coding exons defines exitrons and their role in proteome plasticity.
Marquez, Yamile; H�pfler, Markus; Ayatollahi, Zahra; Barta, Andrea; Kalyna, Maria
Alternative splicing (AS) diversifies transcriptomes and proteomes and is widely recognized as a key mechanism for regulating gene expression. Previously, in an analysis of intron retention events in Arabidopsis, we found unusual AS events inside annotated protein-coding exons. Here, we also identify such AS events in human and use these two sets to analyse their features, regulation, functional impact, and evolutionary origin. As these events involve introns with features of both introns and protein-coding exons, we name them exitrons (exonic introns). Though exitrons were detected as a subset of retained introns, they are clearly distinguishable, and their splicing results in transcripts with different fates. About half of the 1002 Arabidopsis and 923 human exitrons have sizes of multiples of 3 nucleotides (nt). Splicing of these exitrons results in internally deleted proteins and affects protein domains, disordered regions, and various post-translational modification sites, thus broadly impacting protein function. Exitron splicing is regulated across tissues, in response to stress and in carcinogenesis. Intriguingly, annotated intronless genes can be also alternatively spliced via exitron usage. We demonstrate that at least some exitrons originate from ancestral coding exons. Based on our findings, we propose a "splicing memory" hypothesis whereby upon intron loss imprints of former exon borders defined by vestigial splicing regulatory elements could drive the evolution of exitron splicing. Altogether, our studies show that exitron splicing is a conserved strategy for increasing proteome plasticity in plants and animals, complementing the repertoire of AS events. PMID:25934563
Molecular characterization of ten F8 splicing mutations in RNA isolated from patient's leucocytes: assessment of in silico prediction tools accuracy.
Martorell, L; Corrales, I; Ramirez, L; Parra, R; Raya, A; Barquinero, J; Vidal, F
Although 8% of reported FVIII gene (F8) mutations responsible for haemophilia A (HA) affect mRNA processing, very few have been fully characterized at the mRNA level and/or systematically predicted their biological consequences by in silico analysis. This study is aimed to elucidate the effect of potential splice site mutations (PSSM) on the F8 mRNA processing, investigate its correlation with disease severity, and assess their concordance with in silico predictions. We studied the F8 mRNA from 10 HA patient's leucocytes with PSSM by RT-PCR and compared the experimental results with those predicted in silico. The mRNA analysis could explain all the phenotypes observed and demonstrated exon skipping in six cases (c.222G>A, c.601+1delG, c.602-11T>G, c.671-3C>G, c.6115+9C>G and c.6116-1G>A) and activation of cryptic splicing sites, both donor (c.1009+1G>A and c.1009+3A>C) and acceptor sites (c.266-3delC and c.5587-1G>A). In contrast, the in silico analysis was able to predict the score variation of most of the affected splice site, but the precise mechanism could only be correctly determined in two of the 10 mutations analysed. In addition, we have detected aberrant F8 transcripts, even in healthy controls, so this must be taken into account as they could mask the actual contribution of some PSSM. We conclude that F8 mRNA analysis using leucocytes still constitutes an excellent approach to investigate the transcriptional effects of the PSSM in HA, whereas prediction in silico is not always reliable for diagnostic decision-making. PMID:25652415
Genome-Wide Analysis of Heat-Sensitive Alternative Splicing in Physcomitrella patens1[W][OPEN
Chang, Chiung-Yun; Lin, Wen-Dar; Tu, Shih-Long
Plant growth and development are constantly influenced by temperature fluctuations. To respond to temperature changes, different levels of gene regulation are modulated in the cell. Alternative splicing (AS) is a widespread mechanism increasing transcriptome complexity and proteome diversity. Although genome-wide studies have revealed complex AS patterns in plants, whether AS impacts the stress defense of plants is not known. We used heat shock (HS) treatments at nondamaging temperature and messenger RNA sequencing to obtain HS transcriptomes in the moss Physcomitrella patens. Data analysis identified a significant number of novel AS events in the moss protonema. Nearly 50% of genes are alternatively spliced. Intron retention (IR) is markedly repressed under elevated temperature but alternative donor/acceptor site and exon skipping are mainly induced, indicating differential regulation of AS in response to heat stress. Transcripts undergoing heat-sensitive IR are mostly involved in specific functions, which suggests that plants regulate AS with transcript specificity under elevated temperature. An exonic GAG-repeat motif in these IR regions may function as a regulatory cis-element in heat-mediated AS regulation. A conserved AS pattern for HS transcription factors in P. patens and Arabidopsis (Arabidopsis thaliana) reveals that heat regulation for AS evolved early during land colonization of green plants. Our results support that AS of specific genes, including key HS regulators, is fine-tuned under elevated temperature to modulate gene regulation and reorganize metabolic processes. PMID:24777346
Functional implications of the emergence of alternative splicing in hnRNP A/B transcripts
Han, Siew Ping; Kassahn, Karin S.; Skarshewski, Adam; Ragan, Mark A.; Rothnagel, Joseph A.; Smith, Ross
The heterogeneous nuclear ribonucleoproteins (hnRNPs) A/B are a family of RNA-binding proteins that participate in various aspects of nucleic acid metabolism, including mRNA trafficking, telomere maintenance, and splicing. They are both regulators and targets of alternative splicing, and the patterns of alternative splicing of their transcripts have diverged between paralogs and between orthologs in different species. Surprisingly, the extent of this splicing variation and its implications for post-transcriptional regulation have remained largely unexplored. Here, we conducted a detailed analysis of hnRNP A/B sequences and expression patterns across six vertebrates. Alternative exons emerged via the introduction of new splice sites, changes in the strengths of existing splice sites, and the accumulation of auxiliary splicing regulatory motifs. Observed isoform expression patterns could be attributed to the frequency and strength of cis-elements. We found a trend toward increased splicing variation in mammals and identified novel alternatively spliced isoforms in human and chicken. Pulldown and translational assays demonstrated that the inclusion of alternative exons altered the affinity of hnRNP A/B proteins for their cognate nucleic acids and modified protein expression levels. As the hnRNPs A/B regulate several key steps in mRNA processing, the involvement of diverse hnRNP isoforms in multiple cellular contexts and species implies concomitant differences in the transcriptional output of these systems. We conclude that the emergence of alternative splicing in the hnRNPs A/B has contributed to the diversification of their roles in the regulation of alternative splicing and has thus added an unexpected layer of regulatory complexity to transcription in vertebrates. PMID:20651029
Kinetics of membrane-bound nitrate reductase A from Escherichia coli with analogues of physiological electron donors--different reaction sites for menadiol and duroquinol.
Giordani, R; Buc, J; Cornish-Bowden, A; C�rdenas, M L
We have compared the steady-state kinetics of wild-type nitrate reductase A and two mutant forms with altered beta subunits. To mimic conditions in vivo as closely as possible, we used analogues of the physiological quinols as electron donors and membranes with overexpressed nitrate reductase A in preference to a purified alpha beta gamma complex. With the wild-type enzyme both menadiol and duroquinol supply their electrons for the reduction of nitrate at rates that depend on the square of the quinol concentration, menadiol having the higher catalytic constant. The results as a whole are consistent with a substituted-enzyme mechanism for the reduction of nitrate by the quinols. Kinetic experiments suggest that duroquinol and menadiol deliver their electrons at different sites on nitrate reductase, with cross-inhibition. Menadiol inhibits the duroquinol reaction strongly, suggesting that menaquinol may be the preferred substrate in vivo. To examine whether electron transfer from menadiol and duroquinol for nitrate reduction requires the presence of all of the Fe-S centres, we have studied the steady-state kinetics of mutants with beta subunits that lack an Fe-S centre. The loss of the highest-potential Fe-S centre results in an enzyme without menadiol activity, but retaining duroquinol activity; the kinetic parameters are within a factor of two of those of the wild-type enzyme, indicating that this centre is not required for the duroquinol activity. The loss of a low-potential Fe-S centre affects the activity with both quinols: the enzyme is still active but the catalytic constants for both quinols are decreased by about 75%, indicating that this centre is important but not essential for the activity. The existence of a specific site of reaction on nitrate reductase for each quinol, together with the differences in the effects on the two quinols produced by the loss of the Fe-S centre of +80 mV, suggests that the pathways for transfer of electrons from duroquinol and menadiol are not identical. PMID:9428711
Splicing and alternative splicing in rice and humans.
E, Zhiguo; Wang, Lei; Zhou, Jianhua
Rice is a monocot gramineous crop, and one of the most important staple foods. Rice is considered a model species for most gramineous crops. Extensive research on rice has provided critical guidance for other crops, such as maize and wheat. In recent years, climate change and exacerbated soil degradation have resulted in a variety of abiotic stresses, such as greenhouse effects, lower temperatures, drought, floods, soil salinization and heavy metal pollution. As such, there is an extremely high demand for additional research, in order to address these negative factors. Studies have shown that the alternative splicing of many genes in rice is affected by stress conditions, suggesting that manipulation of the alternative splicing of specific genes may be an effective approach for rice to adapt to abiotic stress. With the advancement of microarrays, and more recently, next generation sequencing technology, several studies have shown that more than half of the genes in the rice genome undergo alternative splicing. This mini-review summarizes the latest progress in the research of splicing and alternative splicing in rice, compared to splicing in humans. Furthermore, we discuss how additional studies may change the landscape of investigation of rice functional genomics and genetically improved rice. PMID:24064058
Fatigue and corrosion in aircraft pressure cabin lap splices
R. J. H Wanhill; M. F. J Koolloos
Aircraft structures are susceptible to fatigue and corrosion damage, notably at joints. There may be interactions between fatigue and corrosion, especially as aircraft age. Longitudinal lap splices from several types of transport aircraft pressure cabins were disassembled and investigated for Multiple Site Damage (MSD) fatigue cracking and corrosion. The results are compared with NASA data for a full-scale fatigue test.
Alternative Splicing of RNA Triplets Is Often Regulated and Accelerates Proteome Evolution
Bradley, Robert K.
Thousands of human genes contain introns ending in NAGNAG (N any nucleotide), where both NAGs can function as 3? splice sites, yielding isoforms that differ by inclusion/exclusion of three bases. However, few models exist ...
Therapeutic potential of manipulating VEGF splice isoforms in oncology.
Rennel, Emma S; Harper, Steven J; Bates, David O
Anti-angiogenic therapies currently revolve around targeting vascular endothelial growth factor-A (VEGF-A) or its receptors. These therapies are effective to some degree, but have low response rates and poor side-effect profiles. Part of these problems is likely to be due to their lack of specificity between pro- and anti-angiogenic isoforms, and their nonspecific effects on proactive, pleiotropic survival and maintenance roles of VEGF-A in endothelial and other cell types. An alternative approach, and one which has recently been shown to be effective in animal models of neovascularization in the eye, is to target the mechanisms by which the cell generates pro-angiogenic splice forms of VEGF-A, its receptors and, co-incidentally, by targeting the upstream processes, other oncogenes that have antagonistic splice isoforms. The concept here is to target the splicing mechanisms that control splice site choice in the VEGF-A mRNA. Recent evidence on the pharmacological possibilities of such splice factors is described. PMID:19519209
Alternative Splicing Regulates Targeting of Malate Dehydrogenase in Yarrowia lipolytica
Kabran, Philom�ne; Rossignol, Tristan; Gaillardin, Claude; Nicaud, Jean-Marc; Neuv�glise, C�cile
Alternative pre-mRNA splicing is a major mechanism contributing to the proteome complexity of most eukaryotes, especially mammals. In less complex organisms, such as yeasts, the numbers of genes that contain introns are low and cases of alternative splicing (AS) with functional implications are rare. We report the first case of AS with functional consequences in the yeast Yarrowia lipolytica. The splicing pattern was found to govern the cellular localization of malate dehydrogenase, an enzyme of the central carbon metabolism. This ubiquitous enzyme is involved in the tricarboxylic acid cycle in mitochondria and in the glyoxylate cycle, which takes place in peroxisomes and the cytosol. In Saccharomyces cerevisiae, three genes encode three compartment-specific enzymes. In contrast, only two genes exist in Y. lipolytica. One gene (YlMDH1, YALI0D16753g) encodes a predicted mitochondrial protein, whereas the second gene (YlMDH2, YALI0E14190g) generates the cytosolic and peroxisomal forms through the alternative use of two 3?-splice sites in the second intron. Both splicing variants were detected in cDNA libraries obtained from cells grown under different conditions. Mutants expressing the individual YlMdh2p isoforms tagged with fluorescent proteins confirmed that they localized to either the cytosolic or the peroxisomal compartment. PMID:22368181
Hyperfine and Nuclear Quadrupole Tensors of Nitrogen Donors in the QA Site of Bacterial Reaction Centers: Correlation of the Histidine N? Tensors with Hydrogen Bond Strength
X- and Q-band pulsed EPR spectroscopy was applied to study the interaction of the QA site semiquinone (SQA) with nitrogens from the local protein environment in natural abundance 14N and in 15N uniformly labeled photosynthetic reaction centers of Rhodobacter sphaeroides. The hyperfine and nuclear quadrupole tensors for His-M219 N? and Ala-M260 peptide nitrogen (Np) were estimated through simultaneous simulation of the Q-band 15N Davies ENDOR, X- and Q-band 14,15N HYSCORE, and X-band 14N three-pulse ESEEM spectra, with support from DFT calculations. The hyperfine coupling constants were found to be a(14N) = 2.3 MHz, T = 0.3 MHz for His-M219 N? and a(14N) = 2.6 MHz, T = 0.3 MHz for Ala-M260 Np. Despite that His-M219 N? is established as the stronger of the two H-bond donors, Ala-M260 Np is found to have the larger value of a(14N). The nuclear quadrupole coupling constants were estimated as e2Qq/4h = 0.38 MHz, ? = 0.97 and e2Qq/4h = 0.74 MHz, ? = 0.59 for His-M219 N? and Ala-M260 Np, respectively. An analysis of the available data on nuclear quadrupole tensors for imidazole nitrogens found in semiquinone-binding proteins and copper complexes reveals these systems share similar electron occupancies of the protonated nitrogen orbitals. By applying the Townes�Dailey model, developed previously for copper complexes, to the semiquinones, we find the asymmetry parameter ? to be a sensitive probe of the histidine N?�semiquinone hydrogen bond strength. This is supported by a strong correlation observed between ? and the isotropic coupling constant a(14N) and is consistent with previous computational works and our own semiquinone-histidine model calculations. The empirical relationship presented here for a(14N) and ? will provide an important structural characterization tool in future studies of semiquinone-binding proteins. PMID:25026433
Structure of the human myelin/oligodendrocyte glycoprotein gene and multiple alternative spliced isoforms
Pham-Dinh, D.; Gaspera, D.B.; Dautigny, A. [Universite de Paris (France)] [and others
Myelin/oligodendrocyte glycoprotein (MOG), a special component of the central nervous system localization on the outermost lamellae of mature myelin, is a member of the immunoglobulin superfamily. We report here the organization of the human MOG gene, which spans approximately 17 kb, and the characterization of six MOG mRNA splicing variants. The intron/exon structure of the human MOG gene confirmed the splicing pattern, supporting the hypothesis that mRNA isoforms could arise by alternative splicing of a single gene. In addition to the eight exons coding for the major MOG isoform, the human MOG gene also contains 3` region, a previously unknown alternatively spliced coding exon, VIA. Alternative utilization of two acceptor splicing sites for exon VIII could produce two different C-termini. The nucleotide sequences presented here may be a useful tool to study further possible involvement if the MOG gene in hereditary neurological disorders. 23 refs., 5 figs.
A Combinatorial Code for Splicing Silencing: UAGG and GGGG Motifs
Alternative pre-mRNA splicing is widely used to regulate gene expression by tuning the levels of tissue-specific mRNA isoforms. Few regulatory mechanisms are understood at the level of combinatorial control despite numerous sequences, distinct from splice sites, that have been shown to play roles in splicing enhancement or silencing. Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5?-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19) of the glutamate NMDA R1 receptor (GRIN1) transcript. Disruption of three components of the motif pattern converted the CI cassette into a constitutive exon, while predominant skipping was conferred when the same components were introduced, de novo, into a heterologous constitutive exon. Predominant exon silencing was directed by the motif pattern in the presence of six competing exonic splicing enhancers, and this effect was retained after systematically repositioning the two exonic UAGGs within the CI cassette. In this system, hnRNP A1 was shown to mediate silencing while hnRNP H antagonized silencing. Genome-wide computational analysis combined with RT-PCR testing showed that a class of skipped human and mouse exons can be identified by searches that preserve the sequence and spatial configuration of the UAGG and GGGG motifs. This analysis suggests that the multi-component silencing code may play an important role in the tissue-specific regulation of the CI cassette exon, and that it may serve more generally as a molecular language to allow for intricate adjustments and the coordination of splicing patterns from different genes. PMID:15828859
Interpretation of mRNA splicing mutations in genetic disease: review of the literature and guidelines for information-theoretical analysis
Caminsky, Natasha; Mucaki, Eliseos J.; Rogan, Peter K.
The interpretation of genomic variants has become one of the paramount challenges in the post-genome sequencing era. In this review we summarize nearly 20 years of research on the applications of information theory (IT) to interpret coding and non-coding mutations that alter mRNA splicing in rare and common diseases. We compile and summarize the spectrum of published variants analyzed by IT, to provide a broad perspective of the distribution of deleterious natural and cryptic splice site variants detected, as well as those affecting splicing regulatory sequences. Results for natural splice site mutations can be interrogated dynamically with Splicing Mutation Calculator, a companion software program that computes changes in information content for any splice site substitution, linked to corresponding publications containing these mutations. The accuracy of IT-based analysis was assessed in the context of experimentally validated mutations. Because splice site information quantifies binding affinity, IT-based analyses can discern the differences between variants that account for the observed reduced (leaky) versus abolished mRNA splicing. We extend this principle by comparing predicted mutations in natural, cryptic, and regulatory splice sites with observed deleterious phenotypic and benign effects. Our analysis of 1727 variants revealed a number of general principles useful for ensuring portability of these analyses and accurate input and interpretation of mutations. We offer guidelines for optimal use of IT software for interpretation of mRNA splicing mutations. PMID:25717368
Exon First Nucleotide Mutations in Splicing: Evaluation of In Silico Prediction Tools
Grodeck�, Lucie; Lockerov�, Pavla; Rav?ukov�, Barbora; Buratti, Emanuele; Baralle, Francisco E.; Du�ek, Ladislav; Freiberger, Tom�
Mutations in the first nucleotide of exons (E+1) mostly affect pre-mRNA splicing when found in AG-dependent 3? splice sites, whereas AG-independent splice sites are more resistant. The AG-dependency, however, may be difficult to assess just from primary sequence data as it depends on the quality of the polypyrimidine tract. For this reason, in silico prediction tools are commonly used to score 3? splice sites. In this study, we have assessed the ability of sequence features and in silico prediction tools to discriminate between the splicing-affecting and non-affecting E+1 variants. For this purpose, we newly tested 16 substitutions in vitro and derived other variants from literature. Surprisingly, we found that in the presence of the substituting nucleotide, the quality of the polypyrimidine tract alone was not conclusive about its splicing fate. Rather, it was the identity of the substituting nucleotide that markedly influenced it. Among the computational tools tested, the best performance was achieved using the Maximum Entropy Model and Position-Specific Scoring Matrix. As a result of this study, we have now established preliminary discriminative cut-off values showing sensitivity up to 95% and specificity up to 90%. This is expected to improve our ability to detect splicing-affecting variants in a clinical genetic setting. PMID:24586880
Functional Characterization of the spf/ash Splicing Variation in OTC Deficiency of Mice and Man
Viecelli, Hiu Man; R�fenacht, Veronique; P�rez, Bel�n; Ugarte, Magdalena; H�berle, Johannes; Th�ny, Beat; Desviat, Lourdes Ruiz
The spf/ash mouse model of ornithine transcarbamylase (OTC) deficiency, a severe urea cycle disorder, is caused by a mutation (c.386G>A; p.R129H) in the last nucleotide of exon 4 of the Otc gene, affecting the 5� splice site and resulting in partial use of a cryptic splice site 48 bp into the adjacent intron. The equivalent nucleotide change and predicted amino acid change is found in OTC deficient patients. Here we have used liver tissue and minigene assays to dissect the transcriptional profile resulting from the �spf/ash� mutation in mice and man. For the mutant mouse, we confirmed liver transcripts corresponding to partial intron 4 retention by the use of the c.386+48 cryptic site and to normally spliced transcripts, with exon 4 always containing the c.386G>A (p.R129H) variant. In contrast, the OTC patient exhibited exon 4 skipping or c.386G>A (p.R129H)-variant exon 4 retention by using the natural or a cryptic splice site at nucleotide position c.386+4. The corresponding OTC tissue enzyme activities were between 3-6% of normal control in mouse and human liver. The use of the cryptic splice sites was reproduced in minigenes carrying murine or human mutant sequences. Some normally spliced transcripts could be detected in minigenes in both cases. Antisense oligonucleotides designed to block the murine cryptic +48 site were used in minigenes in an attempt to redirect splicing to the natural site. The results highlight the relevance of in depth investigations of the molecular mechanisms of splicing mutations and potential therapeutic approaches. Notably, they emphasize the fact that findings in animal models may not be applicable for human patients due to the different genomic context of the mutations. PMID:25853564
Functional characterization of the spf/ash splicing variation in OTC deficiency of mice and man.
Rivera-Barahona, Ana; S�nchez-Alcudia, Roc�o; Viecelli, Hiu Man; R�fenacht, Veronique; P�rez, Bel�n; Ugarte, Magdalena; H�berle, Johannes; Th�ny, Beat; Desviat, Lourdes Ruiz
The spf/ash mouse model of ornithine transcarbamylase (OTC) deficiency, a severe urea cycle disorder, is caused by a mutation (c.386G>A; p.R129H) in the last nucleotide of exon 4 of the Otc gene, affecting the 5' splice site and resulting in partial use of a cryptic splice site 48 bp into the adjacent intron. The equivalent nucleotide change and predicted amino acid change is found in OTC deficient patients. Here we have used liver tissue and minigene assays to dissect the transcriptional profile resulting from the "spf/ash" mutation in mice and man. For the mutant mouse, we confirmed liver transcripts corresponding to partial intron 4 retention by the use of the c.386+48 cryptic site and to normally spliced transcripts, with exon 4 always containing the c.386G>A (p.R129H) variant. In contrast, the OTC patient exhibited exon 4 skipping or c.386G>A (p.R129H)-variant exon 4 retention by using the natural or a cryptic splice site at nucleotide position c.386+4. The corresponding OTC tissue enzyme activities were between 3-6% of normal control in mouse and human liver. The use of the cryptic splice sites was reproduced in minigenes carrying murine or human mutant sequences. Some normally spliced transcripts could be detected in minigenes in both cases. Antisense oligonucleotides designed to block the murine cryptic +48 site were used in minigenes in an attempt to redirect splicing to the natural site. The results highlight the relevance of in depth investigations of the molecular mechanisms of splicing mutations and potential therapeutic approaches. Notably, they emphasize the fact that findings in animal models may not be applicable for human patients due to the different genomic context of the mutations. PMID:25853564
Alternative splicing can lead to chaos.
Likhoshvai, Vitaly A; Kogai, Vladislav V; Fadeev, Stanislav I; Khlebodarova, Tamara M
Alternative splicing is a widespread phenomenon in higher eukaryotes, where it serves as a mechanism to increase the functional diversity of proteins. This phenomenon has been described for different classes of proteins, including transcription regulatory proteins. We demonstrated that in the simplest genetic system model the formation of the alternatively spliced isoforms with opposite functions (activators and repressors) could be a cause of transition to chaotic dynamics. Under the simplest genetic system we understand a system consisting of a single gene encoding the structure of a transcription regulatory protein whose expression is regulated by a feedback mechanism. As demonstrated by numerical analysis of the models, if the synthesized isoforms regulate the expression of their own gene acting through different sites and independently of each other, for the generation of chaotic dynamics it is sufficient that the regulatory proteins have a dimeric structure. If regulatory proteins act through one site, the chaotic dynamics is generated in the system only when the repressor protein is either a tetrameric or a higher-dimensional multimer. In this case the activator can be a dimer. It was also demonstrated that if the transcription factor isoforms exhibit either activating or inhibiting activity and are lower-dimensional multimers (< 4), independently of the regulation type the model demonstrates either cyclic or stationary trajectories. PMID:25556917
Spliced human endogenous retroviral HERV-H env transcripts in T-cell leukaemia cell lines and normal leukocytes: alternative splicing pattern of HERV-H transcripts
Mats Lindeskog; Jonas Blomberg
The majority of human endogenous retroviral HERV-H elements in the human genome have large deletions in pol and lack most of env, 5-10% are more or less complete with a potentially immuno- suppressive transmembrane protein-encoding env region. Spliced HERV-H env transcripts were detec- ted in T-cell leukaemia cell lines and lymphocytes from healthy blood donors by using RT-PCR. The transcripts
Aberrant splicing of U12-type introns is the hallmark of ZRSR2 mutant myelodysplastic syndrome.
Madan, Vikas; Kanojia, Deepika; Li, Jia; Okamoto, Ryoko; Sato-Otsubo, Aiko; Kohlmann, Alexander; Sanada, Masashi; Grossmann, Vera; Sundaresan, Janani; Shiraishi, Yuichi; Miyano, Satoru; Thol, Felicitas; Ganser, Arnold; Yang, Henry; Haferlach, Torsten; Ogawa, Seishi; Koeffler, H Phillip
Somatic mutations in the spliceosome gene ZRSR2-located on the X chromosome-are associated with myelodysplastic syndrome (MDS). ZRSR2 is involved in the recognition of 3'-splice site during the early stages of spliceosome assembly; however, its precise role in RNA splicing has remained unclear. Here we characterize ZRSR2 as an essential component of the minor spliceosome (U12 dependent) assembly. shRNA-mediated knockdown of ZRSR2 leads to impaired splicing of the U12-type introns and RNA-sequencing of MDS bone marrow reveals that loss of ZRSR2 activity causes increased mis-splicing. These splicing defects involve retention of the U12-type introns, while splicing of the U2-type introns remain mostly unaffected. ZRSR2-deficient cells also exhibit reduced proliferation potential and distinct alterations in myeloid and erythroid differentiation in vitro. These data identify a specific role for ZRSR2 in RNA splicing and highlight dysregulated splicing of U12-type introns as a characteristic feature of ZRSR2 mutations in MDS. PMID:25586593
Aberrant splicing of U12-type introns is the hallmark of ZRSR2 mutant myelodysplastic syndrome
Madan, Vikas; Kanojia, Deepika; Li, Jia; Okamoto, Ryoko; Sato-Otsubo, Aiko; Kohlmann, Alexander; Sanada, Masashi; Grossmann, Vera; Sundaresan, Janani; Shiraishi, Yuichi; Miyano, Satoru; Thol, Felicitas; Ganser, Arnold; Yang, Henry; Haferlach, Torsten; Ogawa, Seishi; Koeffler, H. Phillip
Somatic mutations in the spliceosome gene ZRSR2 � located on the X chromosome � are associated with myelodysplastic syndrome (MDS). ZRSR2 is involved in the recognition of 3? splice site during the early stages of spliceosome assembly; however, its precise role in RNA splicing has remained unclear. Here, we characterize ZRSR2 as an essential component of the minor spliceosome (U12-dependent) assembly. shRNA mediated knockdown of ZRSR2 leads to impaired splicing of the U12-type introns, and RNA-Sequencing of MDS bone marrow reveals that loss of ZRSR2 activity causes increased mis-splicing. These splicing defects involve retention of the U12-type introns while splicing of the U2-type introns remain mostly unaffected. ZRSR2 deficient cells also exhibit reduced proliferation potential and distinct alterations in myeloid and erythroid differentiation in vitro. These data identify a specific role for ZRSR2 in RNA splicing and highlight dysregulated splicing of U12-type introns as a characteristic feature of ZRSR2 mutations in MDS. PMID:25586593
Unusual Intron Conservation near Tissue-Regulated Exons Found by Splicing Microarrays
Sugnet, Charles W; Srinivasan, Karpagam; Clark, Tyson A; O'Brien, Georgeann; Cline, Melissa S; Wang, Hui; Williams, Alan; Kulp, David; Blume, John E; Haussler, David; Ares, Manuel
Alternative splicing contributes to both gene regulation and protein diversity. To discover broad relationships between regulation of alternative splicing and sequence conservation, we applied a systems approach, using oligonucleotide microarrays designed to capture splicing information across the mouse genome. In a set of 22 adult tissues, we observe differential expression of RNA containing at least two alternative splice junctions for about 40% of the 6,216 alternative events we could detect. Statistical comparisons identify 171 cassette exons whose inclusion or skipping is different in brain relative to other tissues and another 28 exons whose splicing is different in muscle. A subset of these exons is associated with unusual blocks of intron sequence whose conservation in vertebrates rivals that of protein-coding exons. By focusing on sets of exons with similar regulatory patterns, we have identified new sequence motifs implicated in brain and muscle splicing regulation. Of note is a motif that is strikingly similar to the branchpoint consensus but is located downstream of the 5? splice site of exons included in muscle. Analysis of three paralogous membrane-associated guanylate kinase genes reveals that each contains a paralogous tissue-regulated exon with a similar tissue inclusion pattern. While the intron sequences flanking these exons remain highly conserved among mammalian orthologs, the paralogous flanking intron sequences have diverged considerably, suggesting unusually complex evolution of the regulation of alternative splicing in multigene families. PMID:16424921
cDNA cloning reveals a tissue specific expression of alternatively spliced transcripts of the ryanodine receptor type 3 (RyR3) calcium release channel.
Marziali, G; Rossi, D; Giannini, G; Charlesworth, A; Sorrentino, V
Ryanodine receptors (RyRs) are a family of intracellular calcium release channels. Three cDNAs encoding different isoforms of RyR have been identified and cloned. We report the complete sequence of the mink RyR3 cDNA and the characterization of three alternative spliced regions. The first two splicing sites are represented by insertions of five and six amino acids, respectively. The third site is represented by a mutually exclusive splicing. The tissue distribution of the alternatively spliced transcripts revealed a ubiquitous expression of splicing site I and a differential distribution of sites II and III, indicating that a further level of complexity in RyR3 expression may result from alternative splicings in this gene. PMID:8925932
Regulation of alternative splicing by PTB and associated factors.
Spellman, R; Rideau, A; Matlin, A; Gooding, C; Robinson, F; McGlincy, N; Grellscheid, S N; Southby, J; Wollerton, M; Smith, C W J
PTB (polypyrimidine tract-binding protein) is a repressive regulator of alternative splicing. We have investigated the role of PTB in three model alternative splicing systems. In the alpha-actinin gene, PTB represses the SM (smooth muscle) exon by binding to key sites in the polypyrimidine tract. Repressive binding to these sites is assisted by co-operative binding to additional downstream sites. SM exon splicing can be activated by CELF proteins, which also bind co-operatively to interspersed sites and displace PTB from the pyrimidine tract. Exon 11 of PTB pre-mRNA is repressed by PTB in an autoregulatory feedback loop. Exon 11-skipped RNA gets degraded through nonsense-mediated decay. Less than 1% of steady-state PTB mRNA is represented by this isoform, but inhibition of nonsense-mediated decay by RNA interference against Upf1 shows that at least 20% of PTB RNA is consumed by this pathway. This represents a widespread but under-appreciated role of alternative splicing in the quantitative regulation of gene expression, an important addition to its role as a generator of protein isoform diversity. Repression of alpha-tropomyosin exon 3 is an exceptional example of PTB regulation, because repression only occurs at high levels in SM cells, despite the fact that PTB is widely expressed. In this case, a PTB-interacting cofactor, raver1, appears to play an important role. By the use of 'tethering' assays, we have identified discrete domains within both PTB and raver1 that mediate their repressive activities on this splicing event. PMID:15916540
High-risk donors: expanding donor criteria.
Jordan, M L; Shapiro, R; Vivas, C A; Scantlebury, V P; Corry, R J; Randhawa, P; Hakala, T R; Starzl, T E
Advances in the surgical techniques, preservation solutions, and methods for predicting eventual long-term renal function from expanded donors will be critical in allowing precise selection criteria for kidneys for transplantation, resulting in the optimum use of a scarce and precious resource. Until other options such as xenotransplantation or tissue engineering become realistic, the challenge for the millennium will be to identify which donor organs previously considered suboptimal can be safely used to expand the organ donor pool. PMID:10083620
Splice junctions are constrained by protein disorder
Smithers, Ben; Oates, Matt E.; Gough, Julian
We have discovered that positions of splice junctions in genes are constrained by the tolerance for disorder-promoting amino acids in the translated protein region. It is known that efficient splicing requires nucleotide bias at the splice junction; the preferred usage produces a distribution of amino acids that is disorder-promoting. We observe that efficiency of splicing, as seen in the amino-acid distribution, is not compromised to accommodate globular structure. Thus we infer that it is the positions of splice junctions in the gene that must be under constraint by the local protein environment. Examining exonic splicing enhancers found near the splice junction in the gene, reveals that these (short DNA motifs) are more prevalent in exons that encode disordered protein regions than exons encoding structured regions. Thus we also conclude that local protein features constrain efficient splicing more in structure than in disorder. PMID:25934802
Splicing signals in Drosophila: intron size, information content, and consensus sequences.
Mount, S M; Burks, C; Hertz, G; Stormo, G D; White, O; Fields, C
A database of 209 Drosophila introns was extracted from Genbank (release number 64.0) and examined by a number of methods in order to characterize features that might serve as signals for messenger RNA splicing. A tight distribution of sizes was observed: while the smallest introns in the database are 51 nucleotides, more than half are less than 80 nucleotides in length, and most of these have lengths in the range of 59-67 nucleotides. Drosophila splice sites found in large and small introns differ in only minor ways from each other and from those found in vertebrate introns. However, larger introns have greater pyrimidine-richness in the region between 11 and 21 nucleotides upstream of 3' splice sites. The Drosophila branchpoint consensus matrix resembles C T A A T (in which branch formation occurs at the underlined A), and differs from the corresponding mammalian signal in the absence of G at the position immediately preceding the branchpoint. The distribution of occurrences of this sequence suggests a minimum distance between 5' splice sites and branchpoints of about 38 nucleotides, and a minimum distance between 3' splice sites and branchpoints of 15 nucleotides. The methods we have used detect no information in exon sequences other than in the few nucleotides immediately adjacent to the splice sites. However, Drosophila resembles many other species in that there is a discontinuity in A + T content between exons and introns, which are A + T rich. PMID:1508718
PTBP1-dependent Regulation of USP5 Alternative RNA Splicing Plays a Role in Glioblastoma Tumorigenesis
Izaguirre, Daisy I.; Zhu, Wen; Hai, Tao; Cheung, Hannah C.; Krahe, Ralf; Cote, Gilbert J.
Aberrant RNA splicing is thought to play a key role in tumorigenesis. The assessment of its specific contributions is limited by the complexity of information derived from genome-wide array-based approaches. We describe how performing splicing factor-specific comparisons using both tumor and cell line datasets may more readily identify physiologically relevant tumor-specific splicing events. Affymetrix exon array data derived from glioblastoma (GBM) tumor samples with defined PTBP1 levels were compared with data from U251 GBM cells with and without PTBP1 knockdown. This comparison yielded overlapping gene sets that comprised only a minor fraction of each dataset. The identification of a novel GBM-specific splicing event involving the USP5 gene led us to further examine its role in tumorigenesis. In GBM, USP5 generates a shorter isoform 2 through recognition of a 5? splice site within exon 15. Production of the USP5 isoform 2 was strongly correlated with PTBP1 expression in GBM tumor samples and cell lines. Splicing regulation was consistent with the presence of an intronic PTBP1 binding site and could be modulated through antisense targeting of the isoform 2 splice site to force expression of isoform 1 in GBM cells. The forced expression of USP5 isoform 1 in two GBM cell lines inhibited cell growth and migration, implying an important role for USP5 splicing in gliomagenesis. These results support a role for aberrant RNA splicing in tumorigenesis and suggest that changes in relatively few genes may be sufficient to drive the process. PMID:21976412
Computational evidence of NAGNAG alternative splicing in human large intergenic noncoding RNA.
Sun, Xiaoyong; Lin, Simon M; Yan, Xiaoyan
NAGNAG alternative splicing plays an essential role in biological processes and represents a highly adaptable system for posttranslational regulation of gene function. NAGNAG alternative splicing impacts a myriad of biological processes. Previous studies of NAGNAG largely focused on messenger RNA. To the best of our knowledge, this is the first study testing the hypothesis that NAGNAG alternative splicing is also operative in large intergenic noncoding RNA (lincRNA). The RNA-seq data sets from recent deep sequencing studies were queried to test our hypothesis. NAGNAG alternative splicing of human lincRNA was identified while querying two independent RNA-seq data sets. Within these datasets, 31 NAGNAG alternative splicing sites were identified in lincRNA. Notably, most exons of lincRNA containing NAGNAG acceptors were longer than those from protein-coding genes. Furthermore, presence of CAG coding appeared to participate in the splice site selection. Finally, expression of the isoforms of NAGNAG lincRNA exhibited tissue specificity. Together, this study improves our understanding of the NAGNAG alternative splicing in lincRNA. PMID:24995327
Frequent Alternative Splicing of Human Genes
Andrey A. Mironov; James Wildon Fickett; Mikhail S. Gelfand
Alternative splicing can produce variant proteins and expression patterns as different as the products of different genes, yet the prevalence of alternative splicing has not been quantified. Here the spliced alignment algorithm was used to make a first inventory of exon-intron structures of known human genes using EST contigs from the TIGR Human Gene Index. The results on any one
Transition splices and cost comparison
NASA Technical Reports Server (NTRS)
Remedios, M. D.
The development and testing of two designs of transition splices are reported. The design goal was to produce splice terminations that are electrically insulated to withstand the environmental conditions of commercial aircraft and are capable of being repaired and reworked on installed cables with the use of hand tools. In addition, a cost study comparison of FCC vs. RCC is reported. The comparison was made on a basis of 10 aircraft with each vehicle using approximately 100,000 feet of wiring and 2,000 connectors. The results are tabulated for seven different wiring configurations.
Some Characterizations in Splicing Systems
NASA Astrophysics Data System (ADS)
Sarmin, Nor Haniza; Yusof, Yuhani; Wan Heng, Fong
The splitting and recombinant of deoxyribonucleic acid or DNA by specified enzymes using concepts in Formal Language Theory was first mathematically modeled by Head in 1987. This splicing system, S can be presented as a set of initial string I over an alphabet A that acts upon 5' or 3' overhangs of restriction enzymes and can be simply viewed as S = (A, I, B, C). In this paper, a great interest in presenting some relations on certain types of splicing system namely null-context, uniform, simple, semi-simple, semi-null and Sk based on differentiating their rules are given as proposition, corollaries and counterexamples.
Serum albumin binding sites properties in donors and in schizophrenia patients: the study of fluorescence decay of the probe K-35 using S-60 synchrotron pulse excitation
NASA Astrophysics Data System (ADS)
Gryzunov, Yu. A.; Syrejshchikova, T. I.; Komarova, M. N.; Misionzhnik, E. Yu; Uzbekov, M. G.; Molodetskich, A. V.; Dobretsov, G. E.; Yakimenko, M. N.
The properties of serum albumin obtained from donors and from paranoid schizophrenia patients were studied with the fluorescent probe K-35 (N-carboxyphenylimide of dimethylaminonaphthalic acid) and time-resolved fluorescence spectroscopy on the SR beam station of the S-60 synchrotron of the Lebedev Physical Institute. The mean fluorescence quantum yield of K-35 in patients serum was decreased significantly by 25-60% comparing with donors. The analysis of pre-exponential factors of fluorescence decay using "amplitude standard" method has shown that in patient sera the fraction of K-35 molecules bound with albumin and inaccessible to fluorescence quenchers ("bright" K-35 molecules with ?1=8.0�0.4 ns) is 1.2-3 times less than in the donor sera. The fraction of K-35 molecules with partly quenched fluorescence ( ?2=1.44�0.22 ns) was significantly increased in schizophrenia patients. The results obtained suggest that the properties of binding region in serum albumin molecules of acute paranoid schizophrenia patients change significantly.
ERIC Educational Resources Information Center
Miller, Richard E.
The rhetorical advantages and dangers involved in casting the students as "future donors" are explained. The way in which the institutions have to change for casting its students as future donors is described.
... Donor Family Newsroom Minorities Contacting My Donor Family Writing anything can be a challenge. Staring at a ... can take to get started. The process of writing your letter may take some time, but at ...
COMMUNICATION: Alternative splicing and genomic stability
NASA Astrophysics Data System (ADS)
Cahill, Kevin
Alternative splicing allows an organism to make different proteins in different cells at different times, all from the same gene. In a cell that uses alternative splicing, the total length of all the exons is much shorter than in a cell that encodes the same set of proteins without alternative splicing. This economical use of exons makes genes more stable during reproduction and development because a genome with a shorter exon length is more resistant to harmful mutations. Genomic stability may be the reason why higher vertebrates splice alternatively. For a broad class of alternatively spliced genes, a formula is given for the increase in their stability.
Control of alternative splicing by forskolin through hnRNP K during neuronal differentiation.
Cao, Wenguang; Razanau, Aleh; Feng, Dairong; Lobo, Vincent G; Xie, Jiuyong
The molecular basis of cell signal-regulated alternative splicing at the 3' splice site remains largely unknown. We isolated a protein kinase A-responsive ribonucleic acid (RNA) element from a 3' splice site of the synaptosomal-associated protein 25 (Snap25) gene for forskolin-inhibited splicing during neuronal differentiation of rat pheochromocytoma PC12 cells. The element binds specifically to heterogeneous nuclear ribonucleo protein (hnRNP) K in a phosphatase-sensitive way, which directly competes with the U2 auxiliary factor U2AF65, an essential component of early spliceosomes. Transcripts with similarly localized hnRNP K target motifs upstream of alternative exons are enriched in genes often associated with neurological diseases. We show that such motifs upstream of the Runx1 exon 6 also bind hnRNP K, and importantly, hnRNP K is required for forskolin-induced repression of the exon. Interestingly, this exon encodes the peptide domain that determines the switch of the transcriptional repressor/activator activity of Runx1, a change known to be critical in specifying neuron lineages. Consistent with an important role of the target genes in neurons, knocking down hnRNP K severely disrupts forskolin-induced neurite growth. Thus, through hnRNP K, the neuronal differentiation stimulus forskolin targets a critical 3' splice site component of the splicing machinery to control alternative splicing of crucial genes. This also provides a regulated direct competitor of U2AF65 for cell signal control of 3' splice site usage. PMID:22684629
Analysis of SRrp86-regulated alternative splicing
Solis, Amanda S
Previous work led to the hypothesis that SRrp86, a related member of the SR protein superfamily, can interact with and modulate the activity of other SR proteins. Here, we sought to test this hypothesis by examining the effect of changing SRrp86 concentrations on overall alternative splicing patterns. SpliceArrays were used to examine 9,854 splicing events in wild-type cells, cells overexpressing SRrp86, and cells treated with siRNAs to knockdown SRrp86. From among the 500 splicing events exhibiting altered splicing under these conditions, the splicing of c-Jun and I?B? were validated as being regulated by SRrp86 resulting in altered regulation of their downstream targets. In both cases, functionally distinct isoforms were generated that demonstrate the role SRrp86 plays in controlling alternative splicing. PMID:20400856
A novel homozygous splicing mutation in PSAP gene causes metachromatic leukodystrophy in two Moroccan brothers.
Siri, Laura; Rossi, Andrea; Lanza, Federica; Mazzotti, Raffaella; Costa, Anna; Stroppiano, Marina; Gaiero, Alberto; Cohen, Amnon; Biancheri, Roberta; Filocamo, Mirella
Prosaposin (PSAP) gene mutations, affecting saposin B (Sap-B) domain, cause a rare metachromatic leukodystrophy (MLD) variant in which arylsulfatase A (ARSA) activity is normal. To date, only 10 different PSAP mutations have been associated with a total of 18 unrelated MLD patients worldwide. In this study, we report for the first time a family with Moroccan origins in which the proband, presenting with a late-infantile onset of neurological involvement and a brain MRI with the typical tigroid MLD pattern, showed normal values of ARSA activity in the presence of an abnormal pattern of urinary sulfatides. In view of these findings, PSAP gene was analyzed, identifying the newly genomic homozygous c.909 + 1G > A mutation occurring within the invariant GT dinucleotide of the intron 8 donor splice site. Reverse transcriptase-polymerase chain reaction (RT-PCR), showing the direct junction of exon 7 to exon 9, confirmed the skipping of the entire exon 8 (p.Gln260_Lys303) which normally contains two cysteine residues (Cys271 and Cys265) involved in disulfide bridges. Our report provides further evidence that phenotypes of patients with Sap-B deficiency vary widely depending on age of onset, type, and severity of symptoms. Awareness of this rare MLD variant is crucial to prevent delayed diagnosis or misdiagnosis and to promptly provide an accurate genetic counseling, including prenatal diagnosis, to families. PMID:24478108
Spontaneous proton transfer to a conserved intein residue determines on-pathway protein splicing
Pereira, Brian; Shemella, Philip T.; Amitai, Gil; Belfort, Georges; Nayak, Saroj K.; Belfort, Marlene
The discovery of inteins, which are protein-splicing elements, has stimulated interest for various applications in chemical biology, bioseparations, drug delivery, and sensor development. However, for inteins to effectively contribute to these applications, an increased mechanistic understanding of cleavage and splicing reactions is required. While the multi-step chemical reaction that leads to splicing is often explored and utilized, it is not clear how the intein navigates through the reaction space. The sequence of reaction steps must progress in concert in order to yield efficient splicing while minimizing off-pathway cleavage reactions. In this study, we demonstrate that formation of a previously identified branched intermediate is the critical step for determining splicing over cleavage products. By combining experimental assays and quantum mechanical simulations, we identify the electrostatic interactions that are important to the dynamics of the reaction steps. We illustrate, via an animated simulation trajectory, a proton transfer from the first C-terminal extein residue to a conserved aspartate, which synchronizes the multi-step enzymatic reaction that is key to splicing. This work provides new insights into the complex interplay between critical active site residues in the protein splicing mechanism, thereby facilitating biotechnological application while shedding light on multi-step enzyme activity. PMID:21185311
The splicing regulator Sam68 binds to a novel exonic splicing silencer and functions in SMN2 alternative splicing in spinal muscular atrophy
Pedrotti, Simona; Bielli, Pamela; Paronetto, Maria Paola; Ciccosanti, Fabiola; Fimia, Gian Maria; Stamm, Stefan; Manley, James L; Sette, Claudio
Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. An almost identical SMN2 gene is unable to compensate for this deficiency because a single C-to-T transition at position +6 in exon-7 causes skipping of the exon by a mechanism not yet fully elucidated. We observed that the C-to-T transition in SMN2 creates a putative binding site for the RNA-binding protein Sam68. RNA pull-down assays and UV-crosslink experiments showed that Sam68 binds to this sequence. In vivo splicing assays showed that Sam68 triggers SMN2 exon-7 skipping. Moreover, mutations in the Sam68-binding site of SMN2 or in the RNA-binding domain of Sam68 completely abrogated its effect on exon-7 skipping. Retroviral infection of dominant-negative mutants of Sam68 that interfere with its RNA-binding activity, or with its binding to the splicing repressor hnRNP A1, enhanced exon-7 inclusion in endogenous SMN2 and rescued SMN protein expression in fibroblasts of SMA patients. Our results thus indicate that Sam68 is a novel crucial regulator of SMN2 splicing. PMID:20186123
Chaney, John; Suzuki, Yoshikazu; Cantu, Edward
The criteria that define acceptable physiologic and social parameters for lung donation have remained constant since their empiric determination in the 1980s. These criteria include a donor age between 25-40, a arterial partial pressure of oxygen (PaO2)/FiO2 ratio greater than 350, no smoking history, a clear chest X-ray, clean bronchoscopy, and a minimal ischemic time. Due to the paucity of organ donors, and the increasing number of patients requiring lung transplant, finding a donor that meets all of these criteria is quite rare. As such, many transplants have been performed where the donor does not meet these stringent criteria. Over the last decade, numerous reports have been published examining the effects of individual acceptance criteria on lung transplant survival and graft function. These studies suggest that there is little impact of the historical criteria on either short or long term outcomes. For age, donors should be within 18 to 64 years old. Gender may relay benefit to all female recipients especially in male to female transplants, although results are mixed in these studies. Race matched donor/recipients have improved outcomes and African American donors convey worse prognosis. Smoking donors may decrease recipient survival post transplant, but provide a life saving opportunity for recipients that may otherwise remain on the transplant waiting list. No specific gram stain or bronchoscopic findings are reflected in recipient outcomes. Chest radiographs are a poor indicator of lung donor function and should not adversely affect organ usage aside for concerns over malignancy. Ischemic time greater than six hours has no documented adverse effects on recipient mortality and should not limit donor retrieval distances. Brain dead donors and deceased donors have equivalent prognosis. Initial PaO2/FiO2 ratios less than 300 should not dissuade donor organ usage, although recruitment techniques should be implemented with intent to transplant. PMID:25132970
Single-Channel Properties of Recombinant AMPA Receptors Depend on RNA Editing, Splice Variation, and Subunit Composition
Geoffrey T. Swanson; Sunjeev K. Kamboj; Stuart G. Cull-Candy
Non-NMDA glutamate receptor subunits of the AMPA- preferring subfamily combine to form ion channels with heter- ogeneous functional properties. We have investigated the ef- fects of RNA editing at the Q\\/R site, splice variation of the \\
It's a bit over, is that ok? The subtle surplus from tandem alternative splicing.
Szafranski, Karol; Kramer, Marcel
Tandem alternative splice sites (TASS) form a defined class of alternative splicing and give rise to mRNA insertion/deletion variants with only small size differences. Previous work has confirmed evolutionary conservation of TASS elements while many cases show only low tissue specificity of isoform ratios. We pinpoint stochasticity and noise as important methodological issues for the dissection of TASS isoform patterns. Resolving such uncertainties, a recent report showed regulation in a cell culture system, with shifts of alternative splicing isoform ratios dependent on cell density. This novel type of regulation affects not only multiple TASS isoforms, but also other alternative splicing classes, in a concerted manner. Here, we discuss how specific regulatory network architectures may be realized through the novel regulation type and highlight the role of differential isoform functions as a key step in order to better understand the functional role of TASS. PMID:25826565
A novel mechanism for protein-assisted group I intron splicing.
Solem, Amanda; Chatterjee, Piyali; Caprara, Mark G
Previously it was shown that the Aspergillus nidulans (A.n.) mitochondrial COB intron maturase, I-AniI, facilitates splicing of the COB intron in vitro. In this study, we apply kinetic analysis of binding and splicing along with RNA deletion analysis to gain insight into the mechanism of I-AniI facilitated splicing. Our results are consistent with I-AniI and A.n. COB pre-RNA forming a specific but labile encounter complex that is resolved into the native, splicing-competent complex. Significantly, kinetic analysis of splicing shows that the resolution step is rate limiting for splicing. RNA deletion studies show that I-AniI requires most of the A.n. COB intron for binding suggesting that the integrity of the I-AniI-binding site depends on overall RNA tertiary structure. These results, taken together with the observation that A.n. COB intron lacks significant stable tertiary structure in the absence of protein, support a model in which I-AniI preassociates with an unfolded COB intron via a "labile" interaction that facilitates correct folding of the intron catalytic core, perhaps by resolving misfolded RNAs or narrowing the number of conformations sampled by the intron during its search for native structure. The active intron conformation is then "locked in" by specific binding of I-Anil to its intron interaction site. PMID:11991637
Novel Kidins220/ARMS Splice Isoforms: Potential Specific Regulators of Neuronal and Cardiovascular Development
Schmieg, Nathalie; Thomas, Claire; Yabe, Arisa; Lynch, David S.; Iglesias, Teresa; Chakravarty, Probir; Schiavo, Giampietro
Kidins220/ARMS is a transmembrane protein playing a crucial role in neuronal and cardiovascular development. Kidins220/ARMS is a downstream target of neurotrophin receptors and interacts with several signalling and trafficking factors. Through computational modelling, we found two potential sites for alternative splicing of Kidins220/ARMS. The first is located between exon 24 and exon 29, while the second site replaces exon 32 by a short alternative terminal exon 33. Here we describe the conserved occurrence of several Kidins220/ARMS splice isoforms at RNA and protein levels. Kidins220/ARMS splice isoforms display spatio-temporal regulation during development with distinct patterns in different neuronal populations. Neurotrophin receptor stimulation in cortical and hippocampal neurons and neuroendocrine cells induces specific Kidins220/ARMS splice isoforms and alters the appearance kinetics of the full-length transcript. Remarkably, alternative terminal exon splicing generates Kidins220/ARMS variants with distinct cellular localisation: Kidins220/ARMS containing exon 32 is targeted to the plasma membrane and neurite tips, whereas Kidins220/ARMS without exon 33 mainly clusters the full-length protein in a perinuclear intracellular compartment in PC12 cells and primary neurons, leading to a change in neurotrophin receptor expression. Overall, this study demonstrates the existence of novel Kidins220/ARMS splice isoforms with unique properties, revealing additional complexity in the functional regulation of neurotrophin receptors, and potentially other signalling pathways involved in neuronal and cardiovascular development. PMID:26083449
Computational Analysis of an Evolutionarily Conserved VertebrateMuscle Alternative Splicing Program
Das, Debopriya; Clark, Tyson A.; Schweitzer, Anthony; Marr,Henry; Yamamoto, Miki L.; Parra, Marilyn K.; Arribere, Josh; Minovitsky,Simon; Dubchak, Inna; Blume, John E.; Conboy, John G.
A novel exon microarray format that probes gene expression with single exon resolution was employed to elucidate critical features of a vertebrate muscle alternative splicing program. A dataset of 56 microarray-defined, muscle-enriched exons and their flanking introns were examined computationally in order to investigate coordination of the muscle splicing program. Candidate intron regulatory motifs were required to meet several stringent criteria: significant over-representation near muscle-enriched exons, correlation with muscle expression, and phylogenetic conservation among genomes of several vertebrate orders. Three classes of regulatory motifs were identified in the proximal downstream intron, within 200nt of the target exons: UGCAUG, a specific binding site for Fox-1 related splicing factors; ACUAAC, a novel branchpoint-like element; and UG-/UGC-rich elements characteristic of binding sites for CELF splicing factors. UGCAUG was remarkably enriched, being present in nearly one-half of all cases. These studies suggest that Fox and CELF splicing factors play a major role in enforcing the muscle-specific alternative splicing program, facilitating expression of a set of unique isoforms of cytoskeletal proteins that are critical to muscle cell differentiation. Supplementary materials: There are four supplementary tables and one supplementary figure. The tables provide additional detailed information concerning the muscle-enriched datasets, and about over-represented oligonucleotide sequences in the flanking introns. The supplementary figure shows RT-PCR data confirming the muscle-enriched expression of exons predicted from the microarray analysis.
Pre-mRNA splicing is facilitated by an optimal RNA polymerase II elongation rate
Fong, Nova; Kim, Hyunmin; Zhou, Yu; Ji, Xiong; Qiu, Jinsong; Saldi, Tassa; Diener, Katrina; Jones, Ken; Fu, Xiang-Dong
Alternative splicing modulates expression of most human genes. The kinetic model of cotranscriptional splicing suggests that slow elongation expands and that fast elongation compresses the �window of opportunity� for recognition of upstream splice sites, thereby increasing or decreasing inclusion of alternative exons. We tested the model using RNA polymerase II mutants that change average elongation rates genome-wide. Slow and fast elongation affected constitutive and alternative splicing, frequently altering exon inclusion and intron retention in ways not predicted by the model. Cassette exons included by slow and excluded by fast elongation (type I) have weaker splice sites, shorter flanking introns, and distinct sequence motifs relative to �slow-excluded� and �fast-included� exons (type II). Many rate-sensitive exons are misspliced in tumors. Unexpectedly, slow and fast elongation often both increased or both decreased inclusion of a particular exon or retained intron. These results suggest that an optimal rate of transcriptional elongation is required for normal cotranscriptional pre-mRNA splicing. PMID:25452276
RNA Splicing in Plant Mitochondria
Linda Bonen
\\u000a Abstract\\u000a \\u000a Introns within the mitochondrial genes of land plants belong mostly to the group II category of ribozyme mobile retroelements,\\u000a although a minor number (particularly in nonvascular plants) are members of the group I intron family of �homing� ribozymes.\\u000a In vascular plants, the mitochondrial introns often lack conventional structural features, and their degenerate nature is\\u000a correlated with unusual splicing pathways.
Is That Splice Really Good Enough? Improving Fiber Optic Splice Loss Measurement
NEMI Fiber; Optic Splice; J. Meitzler; L. Wesson; P. Arrowsmith; R. Suurmann; M. Rodriguez; D. Gignac; S. Pradhan; J. Garren; J. Johnson; T. Watanabe; E. Mies
Results from a National Electronics Manufacturing Initiative (NEMI) project, formed to improve aspects of fiber optic fusion splicing, are reported. The focus of this paper is ultra low loss splicing for telecommunications product assembly, with typical loss of <0.05 dB per splice for standard SMF-SMF. A detailed review and gap analysis of available industry standards, relevant to splice loss acceptance
Alternative RNA splicing in the nervous system
Paula J. Grabowski; Douglas L. Black
Tissue-specific alternative splicing profoundly effects animal physiology, development and disease, and this is nowhere more evident than in the nervous system. Alternative splicing is a versatile form of genetic control whereby a common pre-mRNA is processed into multiple mRNA isoforms differing in their precise combination of exon sequences. In the nervous system, thousands of alternatively spliced mRNAs are translated into
FOA Lecture 5: Splices and Connectors
NSDL National Science Digital Library
This is the fifth lecture in the FOA series on fiber optics, covering splices and termination. This lecture covers the differences and similarities in splices and connectors and where they are used. Lectures 6 and 7 cover much more detail on fiber optic splices and connectors respectively. Those are available to view separately. Running time for the lecture is 12:21. Flash is required to view the video.
ASD: a bioinformatics resource on alternative splicing
Stefan Stamm; Jean-jack M. Riethoven; Vincent Le Texier; Chellappa Gopalakrishnan; Vasudev Kumanduri; Yesheng Tang; Nuno L. Barbosa-morais; Thangavel Alphonse Thanaraj
Alternative splicing is an important regulatory mechanism of mammalian gene expression. The alternative splicing database (ASD) consortium is systematically collecting and annotating data on alternative splicing. We present the continuation and upgrade of the ASD (T. A. Thanaraj, S. Stamm, F. Clark, J. J. Riethoven, V. Le Texier, J. Muilu (2004) NucleicAcidsRes.32,D64-D69)thatconsistsofcom- putationally and manually generated data. Its largest parts
Nuclear matrix-associated protein SMAR1 regulates alternative splicing via HDAC6-mediated deacetylation of Sam68
Nakka, Kiran Kumar; Chaudhary, Nidhi; Joshi, Shruti; Bhat, Jyotsna; Singh, Kulwant; Chatterjee, Subhrangsu; Malhotra, Renu; De, Abhijit; Santra, Manas Kumar; Dilworth, F. Jeffrey; Chattopadhyay, Samit
Pre-mRNA splicing is a complex regulatory nexus modulated by various trans-factors and their posttranslational modifications to create a dynamic transcriptome through alternative splicing. Signal-induced phosphorylation and dephosphorylation of trans-factors are known to regulate alternative splicing. However, the role of other posttranslational modifications, such as deacetylation/acetylation, methylation, and ubiquitination, that could modulate alternative splicing in either a signal-dependent or -independent manner remain enigmatic. Here, we demonstrate that Scaffold/matrix-associated region-binding protein 1 (SMAR1) negatively regulates alternative splicing through histone deacetylase 6 (HDAC6)-mediated deacetylation of RNA-binding protein Sam68 (Src-associated substrate during mitosis of 68 kDa). SMAR1 is enriched in nuclear splicing speckles and associates with the snRNAs that are involved in splice site recognition. ERK�MAPK pathway that regulates alternative splicing facilitates ERK-1/2�mediated phosphorylation of SMAR1 at threonines 345 and 360 and localizes SMAR1 to the cytoplasm, preventing its interaction with Sam68. We showed that endogenously, SMAR1 through HDAC6 maintains Sam68 in a deacetylated state. However, knockdown or ERK-mediated phosphorylation of SMAR1 releases the inhibitory SMAR1�HDAC6�Sam68 complex, facilitating Sam68 acetylation and alternative splicing. Furthermore, loss of heterozygosity at the Chr.16q24.3 locus in breast cancer cells, wherein the human homolog of SMAR1 (BANP) has been mapped, enhances Sam68 acetylation and CD44 variant exon inclusion. In addition, tail-vein injections in mice with human breast cancer MCF-7 cells depleted for SMAR1 showed increased CD44 variant exon inclusion and concomitant metastatic propensity, confirming the functional role of SMAR1 in regulation of alternative splicing. Thus, our results reveal the complex molecular mechanism underlying SMAR1-mediated signal-dependent and -independent regulation of alternative splicing via Sam68 deacetylation. PMID:26080397
Trans-splicing and operons in metazoans: translational control in maternally regulated development and recovery from growth arrest.
Danks, Gemma B; Raasholm, Martina; Campsteijn, Coen; Long, Abby M; Manak, J Robert; Lenhard, Boris; Thompson, Eric M
Polycistronic mRNAs transcribed from operons are resolved via the trans-splicing of a spliced-leader (SL) RNA. Trans-splicing also occurs at monocistronic transcripts. The phlyogenetically sporadic appearance of trans-splicing and operons has made the driving force(s) for their evolution in metazoans unclear. Previous work has proposed that germline expression drives operon organization in Caenorhabditis elegans, and a recent hypothesis proposes that operons provide an evolutionary advantage via the conservation of transcriptional machinery during recovery from growth arrested states. Using a modified cap analysis of gene expression protocol we mapped sites of SL trans-splicing genome-wide in the marine chordate Oikopleura dioica. Tiled microarrays revealed the expression dynamics of trans-spliced genes across development and during recovery from growth arrest. Operons did not facilitate recovery from growth arrest in O. dioica. Instead, we found that trans-spliced transcripts were predominantly maternal. We then analyzed data from C. elegans and Ciona intestinalis and found that an enrichment of trans-splicing and operon gene expression in maternal mRNA is shared between all three species, suggesting that this may be a driving force for operon evolution in metazoans. Furthermore, we found that the majority of known terminal oligopyrimidine (TOP) mRNAs are trans-spliced in O. dioica and that the SL contains a TOP-like motif. This suggests that the SL in O. dioica confers nutrient-dependent translational control to trans-spliced mRNAs via the TOR-signaling pathway. We hypothesize that SL-trans-splicing provides an evolutionary advantage in species that depend on translational control for regulating early embryogenesis, growth and oocyte production in response to nutrient levels. PMID:25525214
Nuclear matrix-associated protein SMAR1 regulates alternative splicing via HDAC6-mediated deacetylation of Sam68.
Nakka, Kiran Kumar; Chaudhary, Nidhi; Joshi, Shruti; Bhat, Jyotsna; Singh, Kulwant; Chatterjee, Subhrangsu; Malhotra, Renu; De, Abhijit; Santra, Manas Kumar; Dilworth, F Jeffrey; Chattopadhyay, Samit
Pre-mRNA splicing is a complex regulatory nexus modulated by various trans-factors and their posttranslational modifications to create a dynamic transcriptome through alternative splicing. Signal-induced phosphorylation and dephosphorylation of trans-factors are known to regulate alternative splicing. However, the role of other posttranslational modifications, such as deacetylation/acetylation, methylation, and ubiquitination, that could modulate alternative splicing in either a signal-dependent or -independent manner remain enigmatic. Here, we demonstrate that Scaffold/matrix-associated region-binding protein 1 (SMAR1) negatively regulates alternative splicing through histone deacetylase 6 (HDAC6)-mediated deacetylation of RNA-binding protein Sam68 (Src-associated substrate during mitosis of 68 kDa). SMAR1 is enriched in nuclear splicing speckles and associates with the snRNAs that are involved in splice site recognition. ERK-MAPK pathway that regulates alternative splicing facilitates ERK-1/2-mediated phosphorylation of SMAR1 at threonines 345 and 360 and localizes SMAR1 to the cytoplasm, preventing its interaction with Sam68. We showed that endogenously, SMAR1 through HDAC6 maintains Sam68 in a deacetylated state. However, knockdown or ERK-mediated phosphorylation of SMAR1 releases the inhibitory SMAR1-HDAC6-Sam68 complex, facilitating Sam68 acetylation and alternative splicing. Furthermore, loss of heterozygosity at the Chr.16q24.3 locus in breast cancer cells, wherein the human homolog of SMAR1 (BANP) has been mapped, enhances Sam68 acetylation and CD44 variant exon inclusion. In addition, tail-vein injections in mice with human breast cancer MCF-7 cells depleted for SMAR1 showed increased CD44 variant exon inclusion and concomitant metastatic propensity, confirming the functional role of SMAR1 in regulation of alternative splicing. Thus, our results reveal the complex molecular mechanism underlying SMAR1-mediated signal-dependent and -independent regulation of alternative splicing via Sam68 deacetylation. PMID:26080397
30 CFR 57.12088 - Splicing trailing cables.
Code of Federal Regulations, 2010 CFR
...LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES Electricity Underground Only � 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its...
30 CFR 57.12088 - Splicing trailing cables.
Code of Federal Regulations, 2011 CFR
...LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES Electricity Underground Only � 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its...
30 CFR 57.12088 - Splicing trailing cables.
Code of Federal Regulations, 2014 CFR
...LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES Electricity Underground Only � 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its...
30 CFR 57.12088 - Splicing trailing cables.
Code of Federal Regulations, 2012 CFR
...LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES Electricity Underground Only � 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its...
Evolutionary conservation analysis increases the colocalization of predicted exonic splicing enhancers in the BRCA1 gene with missense sequence changes and in-frame deletions, but not polymorphisms
Pettigrew, Christopher; Wayte, Nicola; Lovelock, Paul K; Tavtigian, Sean V; Chenevix-Trench, Georgia; Spurdle, Amanda B; Brown, Melissa A
Introduction Aberrant pre-mRNA splicing can be more detrimental to the function of a gene than changes in the length or nature of the encoded amino acid sequence. Although predicting the effects of changes in consensus 5' and 3' splice sites near intron:exon boundaries is relatively straightforward, predicting the possible effects of changes in exonic splicing enhancers (ESEs) remains a challenge. Methods As an initial step toward determining which ESEs predicted by the web-based tool ESEfinder in the breast cancer susceptibility gene BRCA1 are likely to be functional, we have determined their evolutionary conservation and compared their location with known BRCA1 sequence variants. Results Using the default settings of ESEfinder, we initially detected 669 potential ESEs in the coding region of the BRCA1 gene. Increasing the threshold score reduced the total number to 464, while taking into consideration the proximity to splice donor and acceptor sites reduced the number to 211. Approximately 11% of these ESEs (23/211) either are identical at the nucleotide level in human, primates, mouse, cow, dog and opossum Brca1 (conserved) or are detectable by ESEfinder in the same position in the Brca1 sequence (shared). The frequency of conserved and shared predicted ESEs between human and mouse is higher in BRCA1 exons (2.8 per 100 nucleotides) than in introns (0.6 per 100 nucleotides). Of conserved or shared putative ESEs, 61% (14/23) were predicted to be affected by sequence variants reported in the Breast Cancer Information Core database. Applying the filters described above increased the colocalization of predicted ESEs with missense changes, in-frame deletions and unclassified variants predicted to be deleterious to protein function, whereas they decreased the colocalization with known polymorphisms or unclassified variants predicted to be neutral. Conclusion In this report we show that evolutionary conservation analysis may be used to improve the specificity of an ESE prediction tool. This is the first report on the prediction of the frequency and distribution of ESEs in the BRCA1 gene, and it is the first reported attempt to predict which ESEs are most likely to be functional and therefore which sequence variants in ESEs are most likely to be pathogenic. PMID:16280041
Regulation of Splicing Factors by Alternative Splicing and NMD Is Conserved between Kingdoms Yet Evolutionarily Flexible
Lareau, Liana F.; Brenner, Steven E.
Ultraconserved elements, unusually long regions of perfect sequence identity, are found in genes encoding numerous RNA-binding proteins including arginine-serine rich (SR) splicing factors. Expression of these genes is regulated via alternative splicing of the ultraconserved regions to yield mRNAs that are degraded by nonsense-mediated mRNA decay (NMD), a process termed unproductive splicing (Lareau et al. 2007; Ni et al. 2007). As all human SR genes are affected by alternative splicing and NMD, one might expect this regulation to have originated in an early SR gene and persisted as duplications expanded the SR family. But in fact, unproductive splicing of most human SR genes arose independently (Lareau et al. 2007). This paradox led us to investigate the origin and proliferation of unproductive splicing in SR genes. We demonstrate that unproductive splicing of the splicing factor SRSF5 (SRp40) is conserved among all animals and even observed in fungi; this is a rare example of alternative splicing conserved between kingdoms, yet its effect is to trigger mRNA degradation. As the gene duplicated, the ancient unproductive splicing was lost in paralogs, and distinct unproductive splicing evolved rapidly and repeatedly to take its place. SR genes have consistently employed unproductive splicing, and while it is exceptionally conserved in some of these genes, turnover in specific events among paralogs shows flexible means to the same regulatory end. PMID:25576366
The exon A (C77G) mutation is a common cause of abnormal CD45 splicing in humans.
Tchilian, E Z; Wallace, D L; Imami, N; Liao, H X; Burton, C; Gotch, F; Martinson, J; Haynes, B F; Beverley, P C
The leukocyte common (CD45) Ag is essential for normal T lymphocyte function and alternative splicing at the N terminus of the gene is associated with changes in T cell maturation and differentiation. Recently, a statistically significant association was reported in a large series of human thymus samples between phenotypically abnormal CD45 splicing and the presence of the CC chemokine receptor 5 deletion 32 (CCR5del32) allele, which confers resistance to HIV infection in homozygotes. We show here that abnormal splicing in these thymus samples is associated with the presence of the only established cause of CD45 abnormal splicing, a C77G transversion in exon A. In addition we have examined 227 DNA samples from peripheral blood of healthy donors and find no association between the exon A (C77G) and CCR5del32 mutations. Among 135 PBMC samples, tested by flow cytometric analysis, all those exhibiting abnormal splicing of CD45 also showed the exon A C77G transversion. We conclude that the exon A (C77G) mutation is a common cause of abnormal CD45 splicing and that further disease association studies of this mutation are warranted. PMID:11342634
Molecular aspects of DNA splicing system
NASA Astrophysics Data System (ADS)
Yusof, Yuhani; Lim, Wen Li; Goode, T. Elizabeth; Sarmin, Nor Haniza; Heng, Fong Wan; Wahab, Mohd Firdaus Abd
The pioneer model of deoxyribonucleic acid (DNA) splicing system in a framework of Formal Language Theory was introduced by Head that led to the existence of other models of splicing system, namely Paun, Pixton and Yusof-Goode. These entire models are inspired by the molecular biological process of DNA splicing. Hence, this paper focuses on the translucent DNA splicing process, particularly on the generated language. Starting with some preliminaries in a limit graph, this paper also provides the experimental design with the predicted and actual result.
Novel RNA structural features of an alternatively splicing group II intron from Clostridium tetani
McNeil, Bonnie A.; Zimmerly, Steven
Group II introns are ribozymes in bacterial and organellar genomes that function as self-splicing introns and as retroelements. Previously, we reported that the group II intron C.te.I1 of Clostridium tetani alternatively splices in vivo to produce five distinct coding mRNAs. Accurate fusion of upstream and downstream reading frames requires a shifted 5? splice site located 8 nt upstream of the usual 5? GUGYG motif. This site is specified by the ribozyme through an altered intron/exon-binding site 1 (IBS1�EBS1) pairing. Here we use mutagenesis and self-splicing assays to investigate in more detail the significance of the structural features of the C.te.I1 ribozyme. The shifted 5? splice site is shown to be affected by structures in addition to IBS1�EBS1, and unlike other group II introns, C.te.I1 appears to require a spacer between IBS1 and the GUGYG motif. In addition, the mechanism of 3? exon recognition is modified from the ancestral IIB mechanism to a IIA-like mechanism that appears to be longer than the typical single base-pair interaction and may extend up to 4 bp. The novel ribozyme properties that have evolved for C.te.I1 illustrate the plasticity of group II introns in adapting new structural and catalytic properties that can be utilized to affect gene expression. PMID:24751650
Reduced Mobility of the Alternate Splicing Factor (ASF) through the Nucleoplasm and Steady State Speckle Compartments
Michael J. Kruhlak; Melody A. Lever; Wolfgang Fischle; Eric Verdin; David P. Bazett-Jones; Michael J. Hendzel
Compartmentalization of the nucleus is now recognized as an important level of regulation influenc- ing specific nuclear processes. The mechanism of factor organization and the movement of factors in nuclear space have not been fully determined. Splicing factors, for example, have been shown to move in a directed manner as large intact structures from sites of concen- tration to sites
Four novel cystic fibrosis mutations in splice junction sequences affecting the CFTR nucleotide binding folds.
D�rk, T; Wulbrand, U; T�mmler, B
Single cases of the four novel splice site mutations 1525-1 G-->A (intron 9), 3601-2 A-->G (intron 18), 3850-3 T-->G (intron 19), and 4374 + 1 G-->T (intron 23) were detected in the CFTR gene of cystic fibrosis patients of Indo-Iranian, Turkish, Polish, and German descent. The nucleotide substitutions at the +1, -1, and -2 positions all destroy splice sites and lead to severe disease alleles associated with features typical of gastrointestinal and pulmonary cystic fibrosis disease. The 3850-3 T-to-G change was discovered in a very mildly affected 33-year-old delta F508 compound heterozygote, suggesting that the T-to-G transversion at the less conserved -3 position of the acceptor splice site may retain some wildtype function. PMID:7682196
Trans-splicing versatility of the Ll.LtrB group II intron
Belhocine, Kamila; Mak, Anthony B.; Cousineau, Benoit
Group II introns are found in organelles, bacteria, and archaea. Some harbor an open reading frame (ORF) with reverse transcriptase, maturase, and occasionally endonuclease activities. Group II introns require the assistance of either intron-encoded or free-standing maturases to excise from primary RNA transcripts in vivo. Some ORF-containing group II introns were shown to be mobile retroelements that invade new DNA sites by retrohoming or retrotransposition. Group II introns are also hypothesized to be the ancestors of the spliceosome-dependent nuclear introns and the small nuclear RNAs (snRNAs�U1, U2, U4, U5, and U6) that are part of the spliceosome. The ability of some fragmented group II introns to undergo splicing in trans supports the theory that the snRNAs evolved from portions of group II introns. Here, we developed a Tn5-based genetic screen to explore the trans-splicing potential of the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis. Proficient trans-splicing variants of Ll.LtrB were selected using a highly sensitive trans-splicing/conjugation screen. We report that numerous fragmentation sites located throughout Ll.LtrB support splicing in trans, showing that this intron is remarkably more tolerant to fragmentation than expected from the fragmentation sites uncovered within natural trans-splicing group II introns. This work unveils the great versatility of group II intron fragments to assemble and accurately trans-splice their flanking exons in vivo. PMID:18648072
Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity
Martin, Robert M.; Rino, Jos�; Carvalho, C�lia; Kirchhausen, Tomas; Carmo-Fonseca, Maria
SUMMARY Removal of introns from pre-mRNAs via splicing provides a versatile means of genetic regulation that is often disrupted in human diseases. To decipher how splicing occurs in real time, we have directly examined with single-molecule sensitivity the kinetics of intron excision from pre-mRNA in the nucleus of living human cells. By using two different RNA labeling methods, MS2 and ?N, we show that beta-globin introns are transcribed and excised in 20-30 s. We further show that replacing the weak polypyrimidine (Py) tract in mouse immunoglobulin ? (IgM) pre-mRNA by a U-rich Py decreases the intron lifetime, thus providing direct evidence that splice site strength influences splicing kinetics. We also found that RNA polymerase II transcribes at elongation rates ranging between 3 and 6 kb min-1, and that transcription can be rate limiting for splicing. These results have important implications for mechanistic understanding of co-transcriptional splicing regulation in the live-cell context. PMID:24035393
Functional roles of DExD/H-box RNA helicases in Pre-mRNA splicing.
Liu, Yen-Chi; Cheng, Soo-Chen
Splicing of precursor mRNA takes place via two consecutive steps of transesterification catalyzed by a large ribonucleoprotein complex called the spliceosome. The spliceosome is assembled through ordered binding to the pre-mRNA of five small nuclear RNAs and numerous protein factors, and is disassembled after completion of the reaction to recycle all components. Throughout the splicing cycle, the spliceosome changes its structure, rearranging RNA-RNA, RNA-protein and protein-protein interactions, for positioning and repositioning of splice sites. DExD/H-box RNA helicases play important roles in mediating structural changes of the spliceosome by unwinding of RNA duplexes or disrupting RNA-protein interactions. DExD/H-box proteins are also implicated in the fidelity control of the splicing process at various steps. This review summarizes the functional roles of DExD/H-box proteins in pre-mRNA splicing according to studies conducted mostly in yeast and will discuss the concept of the complicated splicing reaction based on recent findings. PMID:26173448
In silico prediction of splice-altering single nucleotide variants in the human genome
Jian, Xueqiu; Boerwinkle, Eric; Liu, Xiaoming
In silico tools have been developed to predict variants that may have an impact on pre-mRNA splicing. The major limitation of the application of these tools to basic research and clinical practice is the difficulty in interpreting the output. Most tools only predict potential splice sites given a DNA sequence without measuring splicing signal changes caused by a variant. Another limitation is the lack of large-scale evaluation studies of these tools. We compared eight in silico tools on 2959 single nucleotide variants within splicing consensus regions (scSNVs) using receiver operating characteristic analysis. The Position Weight Matrix model and MaxEntScan outperformed other methods. Two ensemble learning methods, adaptive boosting and random forests, were used to construct models that take advantage of individual methods. Both models further improved prediction, with outputs of directly interpretable prediction scores. We applied our ensemble scores to scSNVs from the Catalogue of Somatic Mutations in Cancer database. Analysis showed that predicted splice-altering scSNVs are enriched in recurrent scSNVs and known cancer genes. We pre-computed our ensemble scores for all potential scSNVs across the human genome, providing a whole genome level resource for identifying splice-altering scSNVs discovered from large-scale sequencing studies. PMID:25416802
The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis
Bielli, Pamela; Bus�, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio
Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5? splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival. PMID:24514149
The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis.
Bielli, Pamela; Bus�, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio
Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5' splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival. PMID:24514149
HnRNP A1 controls a splicing regulatory circuit promoting mesenchymal-to-epithelial transition
Bonomi, Serena; di Matteo, Anna; Buratti, Emanuele; Cabianca, Daphne S.; Baralle, Francisco E.; Ghigna, Claudia; Biamonti, Giuseppe
Epithelial-to-mesenchymal transition (EMT) is an embryonic program used by cancer cells to acquire invasive capabilities becoming metastatic. ?Ron, a constitutively active isoform of the Ron tyrosine kinase receptor, arises from skipping of Ron exon 11 and provided the first example of an alternative splicing variant causatively linked to the activation of tumor EMT. Splicing of exon 11 is controlled by two adjacent regulatory elements, a silencer and an enhancer of splicing located in exon 12. The alternative splicing factor and oncoprotein SRSF1 directly binds to the enhancer, induces the production of ?Ron and activates EMT leading to cell locomotion. Interestingly, we now find an important role for hnRNP A1 in controlling the activity of the Ron silencer. HnRNP A1 is able to antagonize the binding of SRSF1 and prevent exon skipping. Notably, hnRNP A1, by inhibiting the production of ?Ron, activates the reversal program, namely the mesenchymal-to-epithelial transition, which instead occurs at the final metastasis sites. Also, hnRNP A1 affects Ron splicing by regulating the expression level of hnRNP A2/B1, which similarly to SRSF1 can promote ?Ron production. These results shed light on how splicing regulation contributes to the tumor progression and provide potential targets to develop anticancer therapies. PMID:23863836
Crystal structures of Aspergillus japonicus fructosyltransferase complex with donor/acceptor substrates reveal complete subsites in the active site for catalysis.
Chuankhayan, Phimonphan; Hsieh, Chih-Yu; Huang, Yen-Chieh; Hsieh, Yi-You; Guan, Hong-Hsiang; Hsieh, Yin-Cheng; Tien, Yueh-Chu; Chen, Chung-De; Chiang, Chien-Min; Chen, Chun-Jung
Fructosyltransferases catalyze the transfer of a fructose unit from one sucrose/fructan to another and are engaged in the production of fructooligosaccharide/fructan. The enzymes belong to the glycoside hydrolase family 32 (GH32) with a retaining catalytic mechanism. Here we describe the crystal structures of recombinant fructosyltransferase (AjFT) from Aspergillus japonicus CB05 and its mutant D191A complexes with various donor/acceptor substrates, including sucrose, 1-kestose, nystose, and raffinose. This is the first structure of fructosyltransferase of the GH32 with a high transfructosylation activity. The structure of AjFT comprises two domains with an N-terminal catalytic domain containing a five-blade beta-propeller fold linked to a C-terminal beta-sandwich domain. Structures of various mutant AjFT-substrate complexes reveal complete four substrate-binding subsites (-1 to +3) in the catalytic pocket with shapes and characters distinct from those of clan GH-J enzymes. Residues Asp-60, Asp-191, and Glu-292 that are proposed for nucleophile, transition-state stabilizer, and general acid/base catalyst, respectively, govern the binding of the terminal fructose at the -1 subsite and the catalytic reaction. Mutants D60A, D191A, and E292A completely lost their activities. Residues Ile-143, Arg-190, Glu-292, Glu-318, and His-332 combine the hydrophobic Phe-118 and Tyr-369 to define the +1 subsite for its preference of fructosyl and glucosyl moieties. Ile-143 and Gln-327 define the +2 subsite for raffinose, whereas Tyr-404 and Glu-405 define the +2 and +3 subsites for inulin-type substrates with higher structural flexibilities. Structural geometries of 1-kestose, nystose and raffinose are different from previous data. All results shed light on the catalytic mechanism and substrate recognition of AjFT and other clan GH-J fructosyltransferases. PMID:20466731
ASAP: the Alternative Splicing Annotation Project
Christopher Lee; Levan Atanelov; Barmak Modrek; Yi Xing
Recently, genomics analyses have demonstrated that alternative splicing is widespread in mammalian genomes (30-60% of genes reported to have multiple isoforms), and maybe one of their most important mechanisms of functional regulation. However, by comparison with other genomics data such as genome annotation, SNPs, or gene expression, there exists relativelylittle database infrastructure for the studyof alternative splicing. We have constructed
The evolving roles of alternative splicing
Liana F Lareau; Richard E Green; Rajiv S Bhatnagar; Steven E Brenner
Alternative splicing is now commonly thought to affect more than half of all human genes. Recent studies have investigated not only the scope but also the biological impact of alternative splicing on a large scale, revealing that its role in generating proteome diversity may be augmented by a role in regulation. For instance, protein function can be regulated by the
Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing
Treutlein, Barbara; Gokce, Ozgun; Quake, Stephen R.; S�dhof, Thomas C.
Neurexins are evolutionarily conserved presynaptic cell-adhesion molecules that are essential for normal synapse formation and synaptic transmission. Indirect evidence has indicated that extensive alternative splicing of neurexin mRNAs may produce hundreds if not thousands of neurexin isoforms, but no direct evidence for such diversity has been available. Here we use unbiased long-read sequencing of full-length neurexin (Nrxn)1?, Nrxn1?, Nrxn2?, Nrxn3?, and Nrxn3? mRNAs to systematically assess how many sites of alternative splicing are used in neurexins with a significant frequency, and whether alternative splicing events at these sites are independent of each other. In sequencing more than 25,000 full-length mRNAs, we identified a novel, abundantly used alternatively spliced exon of Nrxn1? and Nrxn3? (referred to as alternatively spliced sequence 6) that encodes a 9-residue insertion in the flexible hinge region between the fifth LNS (laminin-?, neurexin, sex hormone-binding globulin) domain and the third EGF-like sequence. In addition, we observed several larger-scale events of alternative splicing that deleted multiple domains and were much less frequent than the canonical six sites of alternative splicing in neurexins. All of the six canonical events of alternative splicing appear to be independent of each other, suggesting that neurexins may exhibit an even larger isoform diversity than previously envisioned and comprise thousands of variants. Our data are consistent with the notion that ?-neurexins represent extracellular protein-interaction scaffolds in which different LNS and EGF domains mediate distinct interactions that affect diverse functions and are independently regulated by independent events of alternative splicing. PMID:24639501
Defining the regulatory network of the tissue-specific splicing factors Fox-1 and Fox-2
Zhang, Chaolin; Zhang, Zuo; Castle, John; Sun, Shuying; Johnson, Jason; Krainer, Adrian R.; Zhang, Michael Q.
The precise regulation of many alternative splicing (AS) events by specific splicing factors is essential to determine tissue types and developmental stages. However, the molecular basis of tissue-specific AS regulation and the properties of splicing regulatory networks (SRNs) are poorly understood. Here we comprehensively predict the targets of the brain- and muscle-specific splicing factor Fox-1 (A2BP1) and its paralog Fox-2 (RBM9) and systematically define the corresponding SRNs genome-wide. Fox-1/2 are conserved from worm to human, and specifically recognize the RNA element UGCAUG. We integrate Fox-1/2-binding specificity with phylogenetic conservation, splicing microarray data, and additional computational and experimental characterization. We predict thousands of Fox-1/2 targets with conserved binding sites, at a false discovery rate (FDR) of ?24%, including many validated experimentally, suggesting a surprisingly extensive SRN. The preferred position of the binding sites differs according to AS pattern, and determines either activation or repression of exon recognition by Fox-1/2. Many predicted targets are important for neuromuscular functions, and have been implicated in several genetic diseases. We also identified instances of binding site creation or loss in different vertebrate lineages and human populations, which likely reflect fine-tuning of gene expression regulation during evolution. PMID:18794351
Functional Characterization of Putative Novel Splicing Mutations in the Cardiomyopathy-Causing Genes.
Millat, Gilles; Lafont, Est�le; Nony, S�verine; Rouvet, Isabelle; Bozon, Dominique
Molecular diagnosis of cardiomyopathies remains difficult not only because of the large number of causative genes and the high rate of private mutations but also due to the large number of unclassified variants (UVs) found in patients' DNA. This study reports the functional splicing impact of nine novel genomic variations previously identified in unrelated patients with cardiomyopathies. To identify splice variants among these UVs, a combination of in silico and in vitro hybrid minigene tools was used as transcript is not available. Using this two-step approach, these UVs were reclassified as splicing mutations (MYBPC3-c.655-25A>G, MYBPC3-c.1790G>A (p.Arg597Gln), MYBPC3-c.2414-36G>T) or as mutations with a majority of abnormally spliced transcripts (MYBPC3-c.1182C>A, TNNT2-c.460G>A (p.Glu154Lys), and TNNT2-c.822-3C>A) or as variations with a weak splicing effect (TNNT2-c.1000-38C>A). For the two remaining variations in intron 11 of the TNNT2 gene in the vicinity of the acceptor splice site (c.571-7G>A, c.571-29G>A), a minigene assay was inconclusive as exon 12 is neither recognized as an exon by HeLa nor by H9c2 cells. Our study highlights the importance of the combined use of in silico and in vitro splicing assays to improve the prediction of the functional splicing impact of identified genetic variants if the RNA sample from the patient is not easily available. PMID:25849606
On the effect of nuclear bridge modes on donor-acceptor electronic coupling in donor-bridge-acceptor molecules
NASA Astrophysics Data System (ADS)
Davis, Daly; Toroker, Maytal Caspary; Speiser, Shammai; Peskin, Uri
We report a theoretical study of intra-molecular electronic coupling in a symmetric DBA (donor-bridge-acceptor) complex, in which a donor electronic site is coupled to an acceptor site by way of intervening orbitals of a molecular bridge unit. In the off-resonant (deep tunneling) regime of electronic transport, the lowest unoccupied molecular orbitals (MO's) of the DBA system are split into distinguishable donor/acceptor and bridge orbitals. The effect of geometrical changes at the bridge on the donor/acceptor electronic energy manifold is studied for local stretching and bending modes. It is demonstrated that the energy splitting in the manifold of donor/acceptor unoccupied MOs changes in response to such changes, as assumed in simple McConnell-type models. Limitations of the simple models are revealed where the electronic charging of the bridge orbitals correlates with increasing donor/acceptor orbital energy splitting only for stretching but not for bending bridge modes.
Alcoholism and alternative splicing of candidate genes.
Sasabe, Toshikazu; Ishiura, Shoichi
Gene expression studies have shown that expression patterns of several genes have changed during the development of alcoholism. Gene expression is regulated not only at the level of transcription but also through alternative splicing of pre-mRNA. In this review, we discuss some of the evidence suggesting that alternative splicing of candidate genes such as DRD2 (encoding dopamine D2 receptor) may form the basis of the mechanisms underlying the pathophysiology of alcoholism. These reports suggest that aberrant expression of splice variants affects alcohol sensitivities, and alcohol consumption also regulates alternative splicing. Thus, investigations of alternative splicing are essential for understanding the molecular events underlying the development of alcoholism. PMID:20617039
Residual Strength Analyses of Riveted Lap-Splice Joints
NASA Technical Reports Server (NTRS)
Seshadri, B. R.; Newman, J. C., Jr.
The objective of this paper was to analyze the crack-linkup behavior in riveted-stiffened lap-splice joint panels with small multiple-site damage (MSD) cracks at several adjacent rivet holes. Analyses are based on the STAGS (STructural Analysis of General Shells) code with the critical crack-tip-opening angle (CTOA) fracture criterion. To account for high constraint around a crack front, the "plane strain core" option in STAGS was used. The importance of modeling rivet flexibility with fastener elements that accurately model load transfer across the joint is discussed. Fastener holes are not modeled but rivet connectivity is accounted for by attaching rivets to the sheet on one side of the cracks that simulated both the rivet diameter and MSD cracks. Residual strength analyses made on 2024-T3 alloy (1.6-mm thick) riveted-lap-splice joints with a lead crack and various size MSD cracks were compared with test data from Boeing Airplane Company. Analyses were conducted for both restrained and unrestrained buckling conditions. Comparison of results from these analyses and results from lap-splice-joint test panels, which were partially restrained against buckling indicate that the test results were bounded by the failure loads predicted by the analyses with restrained and unrestrained conditions.
Accurate identification of alternatively spliced exons using support vector machine
Gideon Dror; Rotem Sorek; Ron Shamir
Motivation: Alternative splicing is a major component of the regulation acting on mammalian transcriptomes. It is esti- mated that over half of all human genes have more than one splice variant. Previous studies have shown that alterna- tively spliced exons possess several features that distinguish them from constitutively spliced ones. Recently, we have demonstrated that such features can be used
Skipping of exons by premature termination of transcription and alternative splicing within intron-5 of the sheep SCF gene: a novel splice variant.
Saravanaperumal, Siva Arumugam; Pediconi, Dario; Renieri, Carlo; La Terza, Antonietta
Stem cell factor (SCF) is a growth factor, essential for haemopoiesis, mast cell development and melanogenesis. In the hematopoietic microenvironment (HM), SCF is produced either as a membrane-bound (-) or soluble (+) forms. Skin expression of SCF stimulates melanocyte migration, proliferation, differentiation, and survival. We report for the first time, a novel mRNA splice variant of SCF from the skin of white merino sheep via cloning and sequencing. Reverse transcriptase (RT)-PCR and molecular prediction revealed two different cDNA products of SCF. Full-length cDNA libraries were enriched by the method of rapid amplification of cDNA ends (RACE-PCR). Nucleotide sequencing and molecular prediction revealed that the primary 1519 base pair (bp) cDNA encodes a precursor protein of 274 amino acids (aa), commonly known as 'soluble' isoform. In contrast, the shorter (835 and/or 725 bp) cDNA was found to be a 'novel' mRNA splice variant. It contains an open reading frame (ORF) corresponding to a truncated protein of 181 aa (vs 245 aa) with an unique C-terminus lacking the primary proteolytic segment (28 aa) right after the D(175)G site which is necessary to produce 'soluble' form of SCF. This alternative splice (AS) variant was explained by the complete nucleotide sequencing of splice junction covering exon 5-intron (5)-exon 6 (948 bp) with a premature termination codon (PTC) whereby exons 6 to 9/10 are skipped (Cassette Exon, CE 6-9/10). We also demonstrated that the Northern blot analysis at transcript level is mediated via an intron-5 splicing event. Our data refine the structure of SCF gene; clarify the presence (+) and/or absence (-) of primary proteolytic-cleavage site specific SCF splice variants. This work provides a basis for understanding the functional role and regulation of SCF in hair follicle melanogenesis in sheep beyond what was known in mice, humans and other mammals. PMID:22719917
Interplay between Exonic Splicing Enhancers, mRNA Processing, and mRNA Surveillance in the Dystrophic Mdx Mouse
Buvoli, Massimo; Buvoli, Ada; Leinwand, Leslie A.
Background Pre-mRNA splicing, the removal of introns from RNA, takes place within the spliceosome, a macromolecular complex composed of five small nuclear RNAs and a large number of associated proteins. Spliceosome assembly is modulated by the 5? and 3? splice site consensus sequences situated at the ends of each intron, as well as by exonic and intronic splicing enhancers/silencers recognized by SR and hnRNP proteins. Nonsense mutations introducing a premature termination codon (PTC) often result in the activation of cellular quality control systems that reduce mRNA levels or alter the mRNA splicing pattern. The mdx mouse, a commonly used genetic model for Duchenne muscular dystrophy (DMD), lacks dystrophin by virtue of a premature termination codon (PTC) in exon 23 that also severely reduces the level of dystrophin mRNA. However, the effect of the mutation on dystrophin RNA processing has not yet been described. Methodology/Principal Finding Using combinations of different biochemical and cellular assays, we found that the mdx mutation partially disrupts a multisite exonic splicing enhancer (ESE) that is recognized by a 40 kDa SR protein. In spite of the presence of an inefficient intron 22 3? splice site containing the rare GAG triplet, the mdx mutation does not activate nonsense-associated altered splicing (NAS), but induces exclusively nonsense-mediated mRNA decay (NMD). Functional binding sites for SR proteins were also identified in exon 22 and 24, and in vitro experiments show that SR proteins can mediate direct association between exon 22, 23, and 24. Conclusions/Significance Our findings highlight the complex crosstalk between trans-acting factors, cis-elements and the RNA surveillance machinery occurring during dystrophin mRNA processing. Moreover, they suggest that dystrophin exon�exon interactions could play an important role in preventing mdx exon 23 skipping, as well as in facilitating the pairing of committed splice sites. PMID:17487273
Drosophila ferritin mRNA: alternative RNA splicing regulates the presence of the iron-responsive element
Maria I. Lind; Sophia Ekengren; �jar Melefors; Kenneth S�derh�ll
Several mRNAs encoding the same ferritin subunit of Drosophila melanogaster were identified. Alternative RNA splicing and utilisation of different polyadenylation sites were found to generate the transcripts. The alternative RNA splicing results in ferritin transcripts with four unique 5? untranslated regions. Only one of them contains an iron-responsive element. The iron-responsive element was found to bind in vitro specifically to
Nlend, Rachel Nlend; Meyer, Kathrin
Recent analyses of complete genomes have revealed that alternative splicing became more prevalent and important during eukaryotic evolution. Alternative splicing augments the protein repertoire�particularly that of the human genome�and plays an important role in the development and function of differentiated cell types. However, splicing is also extremely vulnerable, and defects in the proper recognition of splicing signals can give rise to a variety of diseases. In this review, we discuss splicing correction therapies, by using the inherited disease Spinal Muscular Atrophy (SMA) as an example. This lethal early childhood disorder is caused by deletions or other severe mutations of SMN1, a gene coding for the essential survival of motoneurons protein. A second gene copy present in humans and few non-human primates, SMN2, can only partly compensate for the defect because of a single nucleotide change in exon 7 that causes this exon to be skipped in the majority of mRNAs. Thus SMN2 is a prime therapeutic target for SMA. In recent years, several strategies based on small molecule drugs, antisense oligonucleotides or in vivo expressed RNAs have been developed that allow a correction of SMN2 splicing. For some of these, a therapeutic benefit has been demonstrated in mouse models for SMA. This means that clinical trials of such splicing therapies for SMA may become possible in the near future. PMID:20523126
Normal and aberrant splicing of LMNA.
Luo, Yue-Bei; Mastaglia, Frank L; Wilton, Steve D
The LMNA gene gives rise to at least three isoforms (lamin A, C, lamin A?10) as a result of normal alternative splicing, regulated by cis- and trans-acting regulatory factors, as well as the 5' and 3' untranslated regions of the gene. The two main isoforms, lamin A and C, are constitutive components of the fibrous nuclear lamina and have diverse physiological roles, ranging from mechanical nuclear membrane maintenance to gene regulation. The clinical spectrum of diseases (called 'laminopathies') caused by LMNA mutations is broad, including at least eight well-characterised phenotypes, some of which are confined to the skeletal muscles or skin, while others are multisystemic. This review discusses the different alternatively spliced isoforms of LMNA and the regulation of LMNA splicing, as well as the subgroup of mutations that affect splicing of LMNA pre-mRNA, and also seeks to bridge the mis-splicing of LMNA at transcript level and the resulting clinical phenotypes. Finally, we discuss the manipulation of LMNA splicing by splice-switching antisense oligonucleotides and its therapeutic potential for the treatment of some laminopathies. PMID:24459210
RNA-Binding Proteins: Splicing Factors and Disease
Fredericks, Alger M.; Cygan, Kamil J.; Brown, Brian A.; Fairbrother, William G.
Pre-mRNA splicing is mediated by interactions of the Core Spliceosome and an array of accessory RNA binding proteins with cis-sequence elements. Splicing is a major regulatory component in higher eukaryotes. Disruptions in splicing are a major contributor to human disease. One in three hereditary disease alleles are believed to cause aberrant splicing. Hereditary disease alleles can alter splicing by disrupting a splicing element, creating a toxic RNA, or affecting splicing factors. One of the challenges of medical genetics is identifying causal variants from the thousands of possibilities discovered in a clinical sequencing experiment. Here we review the basic biochemistry of splicing, the mechanisms of splicing mutations, the methods for identifying splicing mutants, and the potential of therapeutic interventions. PMID:25985083
Why Selection Might Be Stronger When Populations Are Small: Intron Size and Density Predict within and between-Species Usage of Exonic Splice Associated cis-Motifs
Wu, XianMing; Hurst, Laurence D.
The nearly neutral theory predicts that small effective population size provides the conditions for weakened selection. This is postulated to explain why our genome is more �bloated� than that of, for example, yeast, ours having large introns and large intergene spacer. If a bloated genome is also an error prone genome might it, however, be the case that selection for error-mitigating properties is stronger in our genome? We examine this notion using splicing as an exemplar, not least because large introns can predispose to noisy splicing. We thus ask whether, owing to genomic decay, selection for splice error-control mechanisms is stronger, not weaker, in species with large introns and small populations. In humans much information defining splice sites is in cis-exonic motifs, most notably exonic splice enhancers (ESEs). These act as splice-error control elements. Here then we ask whether within and between-species intron size is a predictor of the commonality of exonic cis-splicing motifs. We show that, as predicted, the proportion of synonymous sites that are ESE-associated and under selection in humans is weakly positively correlated with the size of the flanking intron. In a phylogenetically controlled framework, we observe, also as expected, that mean intron size is both predicted by Ne.? and is a good predictor of cis-motif usage across species, this usage coevolving with splice site definition. Unexpectedly, however, across taxa intron density is a better predictor of cis-motif usage than intron size. We propose that selection for splice-related motifs is driven by a need to avoid decoy splice sites that will be more common in genes with many and large introns. That intron number and density predict ESE usage within human genes is consistent with this, as is the finding of intragenic heterogeneity in ESE density. As intronic content and splice site usage across species is also well predicted by Ne.?, the result also suggests an unusual circumstance in which selection (for cis-modifiers of splicing) might be stronger when population sizes are smaller, as here splicing is noisier, resulting in a greater need to control error-prone splicing. PMID:25771198
Why Selection Might Be Stronger When Populations Are Small: Intron Size and Density Predict within and between-Species Usage of Exonic Splice Associated cis-Motifs.
Wu, XianMing; Hurst, Laurence D
The nearly neutral theory predicts that small effective population size provides the conditions for weakened selection. This is postulated to explain why our genome is more "bloated" than that of, for example, yeast, ours having large introns and large intergene spacer. If a bloated genome is also an error prone genome might it, however, be the case that selection for error-mitigating properties is stronger in our genome? We examine this notion using splicing as an exemplar, not least because large introns can predispose to noisy splicing. We thus ask whether, owing to genomic decay, selection for splice error-control mechanisms is stronger, not weaker, in species with large introns and small populations. In humans much information defining splice sites is in cis-exonic motifs, most notably exonic splice enhancers (ESEs). These act as splice-error control elements. Here then we ask whether within and between-species intron size is a predictor of the commonality of exonic cis-splicing motifs. We show that, as predicted, the proportion of synonymous sites that are ESE-associated and under selection in humans is weakly positively correlated with the size of the flanking intron. In a phylogenetically controlled framework, we observe, also as expected, that mean intron size is both predicted by Ne.? and is a good predictor of cis-motif usage across species, this usage coevolving with splice site definition. Unexpectedly, however, across taxa intron density is a better predictor of cis-motif usage than intron size. We propose that selection for splice-related motifs is driven by a need to avoid decoy splice sites that will be more common in genes with many and large introns. That intron number and density predict ESE usage within human genes is consistent with this, as is the finding of intragenic heterogeneity in ESE density. As intronic content and splice site usage across species is also well predicted by Ne.?, the result also suggests an unusual circumstance in which selection (for cis-modifiers of splicing) might be stronger when population sizes are smaller, as here splicing is noisier, resulting in a greater need to control error-prone splicing. PMID:25771198
Recognition of RNA Branch Point Sequences by the KH Domain of Splicing Factor 1 (Mammalian Branch Point Binding Protein) in a Splicing Factor Complex
Peled-Zehavi, Hadas; Berglund, J. Andrew; Rosbash, Michael; Frankel, Alan D.
Mammalian splicing factor 1 (SF1; also mammalian branch point binding protein [mBBP]; hereafter SF1/mBBP) specifically recognizes the seven-nucleotide branch point sequence (BPS) located at 3? splice sites and participates in the assembly of early spliceosomal complexes. SF1/mBBP utilizes a �maxi-K homology� (maxi-KH) domain for recognition of the single-stranded BPS and requires a cooperative interaction with splicing factor U2AF65 bound to an adjacent polypyrimidine tract (PPT) for high-affinity binding. To investigate how the KH domain of SF1/mBBP recognizes the BPS in conjunction with U2AF and possibly other proteins, we constructed a transcriptional reporter system utilizing human immunodeficiency virus type 1 Tat fusion proteins and examined the RNA-binding specificity of the complex using KH domain and RNA-binding site mutants. We first established that SF1/mBBP and U2AF cooperatively assemble in our reporter system at RNA sites composed of the BPS, PPT, and AG dinucleotide found at 3? splice sites, with endogenous proteins assembled along with the Tat fusions. We next found that the activities of the Tat fusion proteins on different BPS variants correlated well with the known splicing efficiencies of the variants, supporting a model in which the SF1/mBBP-BPS interaction helps determine splicing efficiency prior to the U2 snRNP-BPS interaction. Finally, the likely RNA-binding surface of the maxi-KH domain was identified by mutagenesis and appears similar to that used by �simple� KH domains, involving residues from two putative ? helices, a highly conserved loop, and parts of a ? sheet. Using a homology model constructed from the cocrystal structure of a Nova KH domain-RNA complex (Lewis et al., Cell 100:323�332, 2000), we propose a plausible arrangement for SF1/mBBP-U2AF complexes assembled at 3? splice sites. PMID:11438677
Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells
Palhais, Bruno; Pr�stegaard, Veronica S.; Sabaratnam, Rugivan; Doktor, Thomas Koed; Lutz, Seraina; Burda, Patricie; Suormala, Terttu; Baumgartner, Matthias; Fowler, Brian; Bruun, Gitte Hoffmann; Andersen, Henriette Skovgaard; Ko�ich, Viktor; Andresen, Brage Storstein
The prevalent c.903+469T>C mutation in MTRR causes the cblE type of homocystinuria by strengthening an SRSF1 binding site in an ESE leading to activation of a pseudoexon. We hypothesized that other splicing regulatory elements (SREs) are also critical for MTRR pseudoexon inclusion. We demonstrate that the MTRR pseudoexon is on the verge of being recognized and is therefore vulnerable to several point mutations that disrupt a fine-tuned balance between the different SREs. Normally, pseudoexon inclusion is suppressed by a hnRNP A1 binding exonic splicing silencer (ESS). When the c.903+469T>C mutation is present two ESEs abrogate the activity of the ESS and promote pseudoexon inclusion. Blocking the 3?splice site or the ESEs by SSOs is effective in restoring normal splicing of minigenes and endogenous MTRR transcripts in patient cells. By employing an SSO complementary to both ESEs, we were able to rescue MTRR enzymatic activity in patient cells to approximately 50% of that in controls. We show that several point mutations, individually, can activate a pseudoexon, illustrating that this mechanism can occur more frequently than previously expected. Moreover, we demonstrate that SSO blocking of critical ESEs is a promising strategy to treat the increasing number of activated pseudoexons. PMID:25878036
CDKL5 influences RNA splicing activity by its association to the nuclear speckle molecular machinery.
Ricciardi, Sara; Kilstrup-Nielsen, Charlotte; Bienvenu, Thierry; Jacquette, Aur�lia; Landsberger, Nicoletta; Broccoli, Vania
Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been shown to cause severe neurodevelopmental disorders including infantile spasms, encephalopathy, West-syndrome and an early-onset variant of Rett syndrome. CDKL5 is a serine/threonine kinase whose involvement in Rett syndrome can be inferred by its ability to directly bind and mediate phosphorylation of MeCP2. However, it remains to be elucidated how CDKL5 exerts its function. Here, we report that CDKL5 localizes to specific nuclear foci referred to as nuclear speckles in both cell lines and tissues. These sub-nuclear structures are traditionally considered as storage/modification sites of pre-mRNA splicing factors. Interestingly, we provide evidence that CDKL5 regulates the dynamic behaviour of nuclear speckles. Indeed, CDKL5 overexpression leads to nuclear speckle disassembly, and this event is strictly dependent on its kinase activity. Conversely, its down-regulation affects nuclear speckle morphology leading to abnormally large and uneven speckles. Similar results were obtained for primary adult fibroblasts isolated from CDKL5-mutated patients. Altogether, these findings indicate that CDKL5 controls nuclear speckle morphology probably by regulating the phosphorylation state of splicing regulatory proteins. Nuclear speckles are dynamic sites that can continuously supply splicing factors to active transcription sites, where splicing occurs. Notably, we proved that CDKL5 influences alternative splicing, at least as proved in heterologous minigene assays. In conclusion, we provide evidence that CDKL5 is involved indirectly in pre-mRNA processing, by controlling splicing factor dynamics. These findings identify a biological process whose disregulation might affect neuronal maturation and activity in CDKL5-related disorders. PMID:19740913
Alternative transcription and two modes of splicing result in two myosin light chains from one gene
Yo-Ichi Nabeshima; Yoshiaki Fujii-Kuriyama; Masami Muramatsu; Kikuo Ogata
Chicken skeletal muscle myosin alkali light chains are encoded by a single gene of size 18 kilobases (kb). This gene has two transcription initiation sites from which 17.5- and 8-kb precursor RNAs are transcribed. These RNAs are processed by different modes of splicing to form mRNAs encoding distinct light-chain (LC1 and LC3) proteins.
Functional cystic fibrosis transmembrane conductance regulator expression in cystic fibrosis airway epithelial cells by AAV6.2-mediated segmental trans-splicing.
Song, Yuhu; Lou, Howard H; Boyer, Julie L; Limberis, Maria P; Vandenberghe, Luk H; Hackett, Neil R; Leopold, Philip L; Wilson, James M; Crystal, Ronald G
Cystic fibrosis is characterized by deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) transporter. The packaging constraints of adeno-associated viral (AAV) vectors preclude delivery of both an active promoter and CFTR cDNA to target cells. We hypothesized that segmental trans-splicing, in which two AAV vectors deliver the 5' and 3' halves of the CFTR cDNA, could mediate splicing of two pre-mRNAs into a full-length, functional CFTR mRNA. Using a segmental trans-splicing 5' donor-3' acceptor pair that split the CFTR cDNA between exons 14a and 14b, cotransfection of donor and acceptor plasmids into CFTR(-) cells resulted in full-length CFTR message and protein. Microinjection of plasmids into CFTR(-) cells produced cAMP-activated Cl(-) conductance. Vectors created with an engineered human serotype, AAV6.2, were used to deliver CFTR donor and acceptor constructs, resulting in full-length CFTR mRNA and protein as well as cAMP-activated Cl(-) conductance in CFTR(-) cells, including human CF airway epithelial IB3-1 cells. Thus, segmental trans-splicing can be used with AAV vectors to mediate expression of CFTR, a strategy potentially applicable to individuals with CF. PMID:19257851
Missense mutations in hMLH1 and hMSH2 are associated with exonic splicing enhancers.
Gorlov, Ivan P; Gorlova, Olga Y; Frazier, Marsha L; Amos, Christopher I
There is a critical need to understand why missense mutations are deleterious. The deleterious effects of missense mutations are commonly attributed to their impact on primary amino acid sequence and protein structure. However, several recent studies have shown that some missense mutations are deleterious because they disturb cis-acting splicing elements-so-called "exonic splicing enhancers" (ESEs). It is not clear whether the ESE-related deleterious effects of missense mutations are common. We have evaluated colocalization of pathogenic missense mutations (found in affected individuals) with high-score ESE motifs in the human mismatch-repair genes hMSH2 and hMLH1. We found that pathogenic missense mutations in the hMSH2 and hMLH1 genes are located in ESE sites significantly more frequently than expected. Pathogenic missense mutations also tended to decrease ESE scores, thus leading to a higher propensity for splicing defects. In contrast, nonpathogenic missense mutations (polymorphisms found in unaffected individuals) and nonsense mutations are distributed randomly in relation to ESE sites. Comparison of the observed and expected frequencies of missense mutations in ESE sites shows that pathogenic effects of >/=20% of mutations in hMSH2 result from disruption of ESE sites and disturbed splicing. Similarly, pathogenic effects of >/=16% of missense mutations in the hMLH1 gene are ESE related. The colocalization of pathogenic missense mutations with ESE sites strongly suggests that their pathogenic effects are splicing related. PMID:14526391
Mechanism of alternative splicing and its regulation
Alternative splicing of precursor mRNA is an essential mechanism to increase the complexity of gene expression, and it plays an important role in cellular differentiation and organism development. Regulation of alternative splicing is a complicated process in which numerous interacting components are at work, including cis-acting elements and trans-acting factors, and is further guided by the functional coupling between transcription and splicing. Additional molecular features, such as chromatin structure, RNA structure and alternative transcription initiation or alternative transcription termination, collaborate with these basic components to generate the protein diversity due to alternative splicing. All these factors contributing to this one fundamental biological process add up to a mechanism that is critical to the proper functioning of cells. Any corruption of the process may lead to disruption of normal cellular function and the eventuality of disease. Cancer is one of those diseases, where alternative splicing may be the basis for the identification of novel diagnostic and prognostic biomarkers, as well as new strategies for therapy. Thus, an in-depth understanding of alternative splicing regulation has the potential not only to elucidate fundamental biological principles, but to provide solutions for various diseases. PMID:25798239
FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis
Zhao, Xu; Yang, Ying; Sun, Bao-Fa; Shi, Yue; Yang, Xin; Xiao, Wen; Hao, Ya-Juan; Ping, Xiao-Li; Chen, Yu-Sheng; Wang, Wen-Jia; Jin, Kang-Xuan; Wang, Xing; Huang, Chun-Min; Fu, Yu; Ge, Xiao-Meng; Song, Shu-Hui; Jeong, Hyun Seok; Yanagisawa, Hiroyuki; Niu, Yamei; Jia, Gui-Fang; Wu, Wei; Tong, Wei-Min; Okamoto, Akimitsu; He, Chuan; Danielsen, Jannie M Rendtlew; Wang, Xiu-Jie; Yang, Yun-Gui
The role of Fat Mass and Obesity-associated protein (FTO) and its substrate N6-methyladenosine (m6A) in mRNA processing and adipogenesis remains largely unknown. We show that FTO expression and m6A levels are inversely correlated during adipogenesis. FTO depletion blocks differentiation and only catalytically active FTO restores adipogenesis. Transcriptome analyses in combination with m6A-seq revealed that gene expression and mRNA splicing of grouped genes are regulated by FTO. M6A is enriched in exonic regions flanking 5?- and 3?-splice sites, spatially overlapping with mRNA splicing regulatory serine/arginine-rich (SR) protein exonic splicing enhancer binding regions. Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons. FTO controls exonic splicing of adipogenic regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis. PMID:25412662
Alternative splicing of a group II intron in a surface layer protein gene in Clostridium tetani
McNeil, Bonnie A.; Simon, Dawn M.; Zimmerly, Steven
Group II introns are ribozymes and retroelements found in bacteria, and are thought to have been the ancestors of nuclear pre-mRNA introns. Whereas nuclear introns undergo prolific alternative splicing in some species, group II introns are not known to carry out equivalent reactions. Here we report a group II intron in the human pathogen Clostridium tetani, which undergoes four alternative splicing reactions in vivo. Together with unspliced transcript, five mRNAs are produced, each encoding a distinct surface layer protein isoform. Correct fusion of exon reading frames requires a shifted 5? splice site located 8 nt upstream of the canonical boundary motif. The shifted junction is accomplished by an altered IBS1-EBS1 pairing between the intron and 5? exon. Growth of C. tetani under a variety of conditions did not result in large changes in alternative splicing levels, raising the possibility that alternative splicing is constitutive. This work demonstrates a novel type of gene organization and regulation in bacteria, and provides an additional parallel between group II and nuclear pre-mRNA introns. PMID:24214997
SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice.
Palacino, James; Swalley, Susanne E; Song, Cheng; Cheung, Atwood K; Shu, Lei; Zhang, Xiaolu; Van Hoosear, Mailin; Shin, Youngah; Chin, Donovan N; Keller, Caroline Gubser; Beibel, Martin; Renaud, Nicole A; Smith, Thomas M; Salcius, Michael; Shi, Xiaoying; Hild, Marc; Servais, Rebecca; Jain, Monish; Deng, Lin; Bullock, Caroline; McLellan, Michael; Schuierer, Sven; Murphy, Leo; Blommers, Marcel J J; Blaustein, Cecile; Berenshteyn, Frada; Lacoste, Arnaud; Thomas, Jason R; Roma, Guglielmo; Michaud, Gregory A; Tseng, Brian S; Porter, Jeffery A; Myer, Vic E; Tallarico, John A; Hamann, Lawrence G; Curtis, Daniel; Fishman, Mark C; Dietrich, William F; Dales, Natalie A; Sivasankaran, Rajeev
Spinal muscular atrophy (SMA), which results from the loss of expression of the survival of motor neuron-1 (SMN1) gene, represents the most common genetic cause of pediatric mortality. A duplicate copy (SMN2) is inefficiently spliced, producing a truncated and unstable protein. We describe herein a potent, orally active, small-molecule enhancer of SMN2 splicing that elevates full-length SMN protein and extends survival in a severe SMA mouse model. We demonstrate that the molecular mechanism of action is via stabilization of the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 small nuclear ribonucleic protein (snRNP) complex. The binding affinity of U1 snRNP to the 5' splice site is increased in a sequence-selective manner, discrete from constitutive recognition. This new mechanism demonstrates the feasibility of small molecule-mediated, sequence-selective splice modulation and the potential for leveraging this strategy in other splicing diseases. PMID:26030728
Cisplatin inhibits protein splicing, suggesting inteins as therapeutic targets in mycobacteria.
Zhang, Liyun; Zheng, Yuchuan; Callahan, Brian; Belfort, Marlene; Liu, Yangzhong
Mycobacterium tuberculosis harbors three protein splicing elements, called inteins, in critical genes and their protein products. Post-translational removal of the inteins occurs autocatalytically and is required for function of the respective M. tuberculosis proteins. Inteins are therefore potential targets for antimycobacterial agents. In this work, we report that the splicing activity of the intein present in the RecA recombinase of M. tuberculosis is potently inhibited by the anticancer drug cisplatin (cis-diamminedichloro-platinum(II)). This previously unrecognized activity of cisplatin was established using both an in vitro intein splicing assay, which yielded an IC(50) of ?2 ?M, and a genetic reporter for intein splicing in Escherichia coli. Testing of related platinum(II) complexes indicated that the inhibition activity is highly structure-dependent, with cisplatin exhibiting the best inhibitory effect. Finally, we report that cisplatin is toxic toward M. tuberculosis with a minimum inhibitory concentration of ?40 ?M, and in genetic experiments conducted with the related Mycobacterium bovis bacillus Calmette-Gu�rrin (BCG) strain, we show that cisplatin toxicity can be mitigated by intein overexpression. We propose that cisplatin inhibits intein activity by modifying at least one conserved cysteine residue that is required for splicing. Together these results identify a novel active site inhibitor of inteins and validate inteins as viable targets for small molecule inhibition in mycobacteria. PMID:21059649
Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins
Huelga, Stephanie C.; Vu, Anthony Q.; Arnold, Justin D.; Liang, Tiffany Y.; Liu, Patrick P.; Yan, Bernice Y.; Donohue, John Paul; Shiue, Lily; Hoon, Shawn; Brenner, Sydney; Ares, Manuel; Yeo, Gene W.
SUMMARY Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS) in human cells. Using splicing-sensitive microarrays, cross-linking and immunoprecipitation coupled with high-throughput sequencing, and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins, and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and auto-regulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells. PMID:22574288
Trans-acting factors regulate the expression of CD44 splice variants.
Konig, H; Moll, J; Ponta, H; Herrlich, P
Variant isoforms of the cell surface glycoprotein CD44 (CD44v) are expressed during development, in selected adult tissues and in certain metastatic tumor cells. CD44v differ from the standard isoform (CD44s) by up to ten additional exon sequences included by alternative splicing. By cell fusion experiments, we have obtained evidence for the existence of cell-type specific trans-acting factors recruiting CD44 variant exon sequences. Stable cell hybrids of CD44s and CD44v expressing cells indicated a dominant mechanism for variant-exon inclusion. In transient interspecies heterokaryons of human keratinocytes and rat fibroblasts, the ability of the keratinocytes to include all variant exon sequences in CD44 was conferred completely on the rat fibroblast nucleus. Fusions of cells with complex CD44 splice patterns do not permit interpretation of splice control by the relative abundance of a single trans-acting factor, but rather by (a) positively acting factor(s) recruiting variant exon sequences in the 3' to 5' direction and additional factors selecting individual exons. Since the pancreatic carcinoma cell line BSp73ASML (in contrast to the cervix carcinoma cell lines SiHa and ME180) could not transfer its specific splice pattern in cell fusions, we conclude that in some tumors, splicing is also controlled by mutation of cis-acting recognition sites. Images PMID:8670907
Sex-lethal interacts with splicing factors in vitro and in vivo.
Deshpande, G; Samuels, M E; Schedl, P D
The Drosophila sex determination gene Sex-lethal controls its own expression and the expression of downstream target genes such as transformer by regulating RNA splicing. Genetic and molecular studies have established that Sxl requires the product of another gene, snf, to autoregulate the splicing of its own transcripts. snf has recently been shown to encode a Drosophila U1 and U2 small nuclear ribonucleoprotein particle protein. In the work reported here, we demonstrate that the Sxl and Snf proteins can interact directly in vitro and that these two proteins are part of an RNase-sensitive complex in vivo which can be immunoprecipitated with the Sxl antibody. Unlike bulk Snf protein, which sediments slowly in sucrose gradients, the Snf protein associated with Sxl is in a large, rapidly sedimenting complex. Detailed characterization of the Sxl-Snf complexes from cross-linked extracts indicates that these complexes contain additional small nuclear ribonucleoprotein particle proteins and the U1 and U2 small nuclear RNAs. Finally, consistent with the RNase sensitivity of the Sxl-Snf complexes, Sxl transcripts can also be immunoprecipitated by Sxl antibodies. On the basis of the physical interactions between Sxl and Snf, we present a model for Sxl splicing regulation. This model helps explain how the Sxl protein is able to promote the sex-specific splicing of Sxl transcripts, utilizing target sequences that are distant from the regulated splice sites. PMID:8756662
Localization of an exonic splicing enhancer responsible for mammalian natural trans-splicing
Concha Caudevilla; Carles Codony; Dolors Serra; Guillem Plasencia; Ruth Rom�n; Adolf Graessmann; Guillermina Asins; Montserrat Bach-Elias; Fausto G. Hegardt
Carnitine octanoyltransferase (COT) produces three different transcripts in rat through cis -a ndtrans- splicing reactions, which may lead to the synthesis of two proteins. Generation of the three COT tran- scripts in rat does not depend on sex, development, fat feeding, the inclusion of the peroxisome prolifer- ator diethylhexyl phthalate in the diet or hyper- insulinemia. In addition, trans-splicing was
The behavior of bonded doubler splices for composite sandwich panels
NASA Technical Reports Server (NTRS)
Zeller, T. A.; Weisahaar, T. A.
The results of an investigation into the behavior of adhesively bonded doubler splices of two composite material sandwich panels are presented. The splices are studied from three approaches: analytical; numerical (finite elements); and experimental. Several parameters that characterize the splice are developed to determine their influence upon joint strength. These parameters are: doubler overlap length; core stiffness; laminate bending stiffness; the size of the gap between the spliced sandwich panels; and room and elevated temperatures. Similarities and contrasts between these splices and the physically similar single and double lap joints are discussed. The results of this investigation suggest several possible approaches to improving the strength of the sandwich splices.
Role of Pnn in alternative splicing of a specific subset of lncRNAs of the corneal epithelium
Joo, Jeong Hoon; Ryu, Danny; Peng, Qian
Purpose: GG-H whole transcriptome array analysis suggested involvement of PININ (PNN) in the alternative splicing of multiple long non-coding RNAs (lncRNAs). To further investigate PNN�s role in regulating the alternative splicing of lncRNAs in a corneal epithelial context, we performed detailed analyses for detecting and identifying alternatively spliced lncRNAs. Methods: Total RNA was isolated from PNN knockdown human corneal epithelial (HCET) cells or Pnn-deficient mouse corneas, and subjected to real-time�PCR (RT�PCR) assays, and the alternatively spliced lncRNAs were counted. Alternatively spliced lncRNAs were detected with in situ hybridization with variant-specific RNA probes on human cornea sections. Results: Our analysis uncovered PNN�s impact on the transcript levels of several lncRNAs including Linc00085 and HAS2-AS1. Interestingly, a mouse ortholog of HAS2-AS1, Has2as, clearly exhibited a differential splicing pattern among three major splice variants in the Pnn-deficient mouse cornea. The sequence analyses and quantification of splice variants of candidate lncRNAs, including RP11-295B20.2, RP11�18I14.1, and RP11�322M19.1, demonstrated complex configuration of their splicing changes, with a significant impact of PNN on the process. Knockdown of PNN in HCET cells led to specific changes in the inclusion of multiple cassette exons as well as in the use of alternative splice sites in RP11�322M19.1 and RP11�18I14.1, resulting in considerable net changes in the ratio between the splice variants. Finally, in situ hybridization analyses revealed the presence of RP11�295G20.2 in the nuclei of corneal epithelial cells, but not in the stromal cells of the human cornea, while RP11�322M19.1 was present in epithelial and non-epithelial cells. Conclusions: The data suggest PNN�s role in the alternative splicing of a specific subset of lncRNAs might have a significant impact on the corneal epithelium. PMID:25489234
ERIC Educational Resources Information Center
Schaffhauser, Dian
When times get tough, grown children often turn to their parents for help--for some extra cash, even somewhere to stay. For colleges and universities, that role is filled by alumni donors. In 2011, with education budgets slashed across the country, giving accounted for 6.5 percent of college expenditures, according to the Council for Aid to�
Identifying Potential Kidney Donors Using Social Networking Websites
Chang, Alexander; Anderson, Emily E.; Turner, Hang T.; Shoham, David; Hou, Susan H.; Grams, Morgan
Social networking sites like Facebook may be a powerful tool for increasing rates of live kidney donation. They allow for wide dissemination of information and discussion, and could lessen anxiety associated with a face-to-face request for donation. However, sparse data exist on the use of social media for this purpose. We searched Facebook, the most popular social networking site, for publicly available English-language pages seeking kidney donors for a specific individual, abstracting information on the potential recipient, characteristics of the page itself, and whether potential donors were tested. In the 91 pages meeting inclusion criteria, the mean age of potential recipients was 37 (range: 2�69); 88% were U.S. residents. Other posted information included the individual�s photograph (76%), blood type (64%), cause of kidney disease (43%), and location (71%). Thirty-two percent of pages reported having potential donors tested, and 10% reported receiving a live donor kidney transplant. Those reporting donor testing shared more potential recipient characteristics, provided more information about transplantation, and had higher page traffic. Facebook is already being used to identify potential kidney donors. Future studies should focus on how to safely, ethically, and effectively use social networking sites to inform potential donors and potentially expand live kidney donation. PMID:23600791
Catalytic Key Amino Acids and UDP-Sugar Donor Specificity of a Plant Glucuronosyltransferase, UGT94B1: Molecular Modeling Substantiated by Site-Specific Mutagenesis and Biochemical Analyses1[C][OA
Osmani, Sarah A.; Bak, S�ren; Imberty, Anne; Olsen, Carl Erik; M�ller, Birger Lindberg
The plant UDP-dependent glucosyltransferase (UGT) BpUGT94B1 catalyzes the synthesis of a glucuronosylated cyanidin-derived flavonoid in red daisy (Bellis perennis). The functional properties of BpUGT94B1 were investigated using protein modeling, site-directed mutagenesis, and analysis of the substrate specificity of isolated wild-type and mutated forms of BpUGT94B1. A single unique arginine residue (R25) positioned outside the conserved plant secondary product glycosyltransferase region was identified as crucial for the activity with UDP-glucuronic acid. The mutants R25S, R25G, and R25K all exhibited only 0.5% to 2.5% of wild-type activity with UDP-glucuronic acid, but showed a 3-fold increase in activity with UDP-glucose. The model of BpUGT94B1 also enabled identification of key residues in the acceptor pocket. The mutations N123A and D152A decreased the activity with cyanidin 3-O-glucoside to less than 15% of wild type. The wild-type enzyme activity toward delphinidin-3-O-glucoside was only 5% to 10% of the activity with cyanidin 3-O-glucoside. Independent point mutations of three residues positioned near the acceptor B ring were introduced to increase the activity toward delphinidin-3-O-glucoside. In all three mutant enzymes, the enzymatic activity toward both acceptors was reduced to less than 15% of wild type. The model of BpUGT94B1 allowed for correct identification of catalytically important residues, within as well as outside the plant secondary product glycosyltransferase motif, determining sugar donor and acceptor specificity. PMID:18829982
Catalytic key amino acids and UDP-sugar donor specificity of a plant glucuronosyltransferase, UGT94B1: molecular modeling substantiated by site-specific mutagenesis and biochemical analyses.
Osmani, Sarah A; Bak, S�ren; Imberty, Anne; Olsen, Carl Erik; M�ller, Birger Lindberg
The plant UDP-dependent glucosyltransferase (UGT) BpUGT94B1 catalyzes the synthesis of a glucuronosylated cyanidin-derived flavonoid in red daisy (Bellis perennis). The functional properties of BpUGT94B1 were investigated using protein modeling, site-directed mutagenesis, and analysis of the substrate specificity of isolated wild-type and mutated forms of BpUGT94B1. A single unique arginine residue (R25) positioned outside the conserved plant secondary product glycosyltransferase region was identified as crucial for the activity with UDP-glucuronic acid. The mutants R25S, R25G, and R25K all exhibited only 0.5% to 2.5% of wild-type activity with UDP-glucuronic acid, but showed a 3-fold increase in activity with UDP-glucose. The model of BpUGT94B1 also enabled identification of key residues in the acceptor pocket. The mutations N123A and D152A decreased the activity with cyanidin 3-O-glucoside to less than 15% of wild type. The wild-type enzyme activity toward delphinidin-3-O-glucoside was only 5% to 10% of the activity with cyanidin 3-O-glucoside. Independent point mutations of three residues positioned near the acceptor B ring were introduced to increase the activity toward delphinidin-3-O-glucoside. In all three mutant enzymes, the enzymatic activity toward both acceptors was reduced to less than 15% of wild type. The model of BpUGT94B1 allowed for correct identification of catalytically important residues, within as well as outside the plant secondary product glycosyltransferase motif, determining sugar donor and acceptor specificity. PMID:18829982
Genomics of alternative splicing: evolution, development and pathophysiology.
Gamazon, Eric R; Stranger, Barbara E
Alternative splicing is a major cellular mechanism in metazoans for generating proteomic diversity. A large proportion of protein-coding genes in multicellular organisms undergo alternative splicing, and in humans, it has been estimated that nearly 90 % of protein-coding genes-much larger than expected-are subject to alternative splicing. Genomic analyses of alternative splicing have illuminated its universal role in shaping the evolution of genomes, in the control of developmental processes, and in the dynamic regulation of the transcriptome to influence phenotype. Disruption of the splicing machinery has been found to drive pathophysiology, and indeed reprogramming of aberrant splicing can provide novel approaches to the development of molecular therapy. This review focuses on the recent progress in our understanding of alternative splicing brought about by the unprecedented explosive growth of genomic data and highlights the relevance of human splicing variation on disease and therapy. PMID:24378600
U1 small nuclear RNAs with altered specificity can be stably expressed in mammalian cells and promote permanent changes in pre-mRNA splicing.
Cohen, J B; Broz, S D; Levinson, A D
Pre-mRNA 5' splice site activity depends, at least in part, on base complementarity to U1 small nuclear RNA. In transient coexpression assays, defective 5' splice sites can regain activity in the presence of U1 carrying compensatory changes, but it is unclear whether such mutant U1 RNAs can be permanently expressed in mammalian cells. We have explored this issue to determine whether U1 small nuclear RNAs with altered specificity may be of value to rescue targeted mutant genes or alter pre-mRNA processing profiles. This effort was initiated following our observation that U1 with specificity for a splice site associated with an alternative H-ras exon substantially reduced the synthesis of the potentially oncogenic p21ras protein in transient assays. We describe the development of a mammalian complementation system that selects for removal of a splicing-defective intron placed within a drug resistance gene. Complementation was observed in proportion to the degree of complementarity between transfected mutant U1 genes and different defective splice sites, and all cells selected in this manner were found to express mutant U1 RNA. In addition, these cells showed specific activation of defective splice sites presented by an unlinked reporter gene. We discuss the prospects of this approach to permanently alter the expression of targeted genes in mammalian cells. Images PMID:7682651
Quantification of type II procollagen splice forms using Alternative Transcript-qPCR (AT-qPCR)
McAlinden, Audrey; Shim, Kyu-Hwan; Wirthlin, Louisa; Ravindran, Soumya; Hering, Thomas M.
During skeletal development, the onset of chondrogenic differentiation is marked by expression of the ?1(II) procollagen Col2a1) gene. Exon 2 of Col2a1 codes for a cysteine-rich von Willebrand factor C-like domain. Chondroprogenitors express the exon 2-containing IIA and IID splice forms by utilizing adjacent 5? splice sites separated by 3 base pairs. There is a shift to expression of the shorter, exon 2-lacking IIB splice form with further differentiation. Alternative splicing analysis of Col2a1 splice forms has often relied upon semi-quantitative PCR, using a single set of PCR primers to amplify multiple splice forms. We show that this widely used method is inaccurate due to mismatched amplification efficiency of different-sized PCR products. We have developed the TaqMan�-based AT-qPCR (Alternative Transcript-qPCR) assay to more accurately quantify alternatively spliced mRNA, and demonstrate the measurement of Col2a1 splice form expression in differentiating ATDC5 cells in vitro and in wild type mouse embryonic and postnatal cartilage in vivo. The AT-qPCR assay is based on the use of a multiple amplicon standard (MAS) plasmid, containing a chemically synthesized cluster of splice site-spanning PCR amplicons, to quantify alternative splice forms by standard curve-based qPCR. The MAS plasmid designed for Col2a1 also contained an 18S rRNA amplicon for sample normalization, and an amplicon corresponding to a region spanning exon 52-53 to measure total Col2a1 mRNA. In mouse E12.5 to P70 cartilage, we observed the expected switch between the IIA and IIB splice forms; no IID or IIC splice products were observed. However, in the ATDC5 cultures, predominant expression of the IIA and IID splice forms was found at all times in culture. Additionally, we observed that the sum of the IIA, IIB and IID splice forms comprises only a small fraction of Col2a1 transcripts containing the constitutive exon 52-53 junction. We conclude from our results that the majority of ATDC5 cells in the assay described in this study remained as chondroprogenitors during culture in standard differentiation conditions, and that additional Col2a1 transcripts may be present. The validity of this novel AT-qPCR assay was confirmed by demonstrating the expected Col2a1 isoform expression patterns in vivo in developing mouse cartilage. The ability to measure true levels of procollagen type II splice forms will provide better monitoring of chondrocyte differentiation in other model systems. In addition, the AT-qPCR assay described here could be applied to any gene of interest to detect and quantify known and predicted alternative splice forms and can be scaled up for high throughput assays. PMID:22974592
Insertion loss measurement of low loss fiber optic splices
L. Wesson; P. Arrowsmith; R. Suurmann; D. Gignac; S. Pradhan; J. Garren; T. Watanabe; E. Mies
Results from a National Electronics Manufacturing Initiative (NEMI) project, formed to improve aspects of fiber optic fusion splicing, are reported. The focus of this paper is ultra low loss splicing for telecommunications product assembly, with typical loss of <0.05 dB per splice for standard SMF-SMF. A detailed review of available industry standards, relevant to splice loss acceptance criteria and loss
Highly Conserved Histidine Plays a Dual Catalytic Role in Protein Splicing: a pKa Shift Mechanism
Du, Zhenming; Shemella, Philip T.; Liu, Yangzhong; McCallum, Scott A.; Pereira, Brian; Nayak, Saroj K.; Belfort, Georges; Belfort, Marlene; Wang, Chunyu
Protein splicing is a precise auto-catalytic process in which an intein excises itself from a precursor with the concomitant ligation of the flanking sequences. Protein splicing occurs through acid-base catalysis in which the ionization states of active site residues are crucial to the reaction mechanism. In inteins, several conserved histidines have been shown to play important roles in protein splicing, including the most conserved �B-block� histidine. In this study, we have combined NMR pKa determination with quantum mechanics/molecular mechanics (QM/MM) modeling to study engineered inteins from Mycobacterium tuberculosis (Mtu) RecA intein. We demonstrate a dramatic pKa shift for the invariant B-block histidine, the most conserved residue among inteins. The B-block histidine has a pKa of 7.3 � 0.6 in a precursor and a pKa of < 3.5 in a spliced intein. The pKa values and QM/MM data suggest that the B-block histidine has a dual role in the acid-base catalysis of protein splicing. This histidine likely acts as a general base to initiate splicing with an acyl shift and then as a general acid to cause the breakdown of the scissile bond. The proposed pKa shift mechanism accounts for the biochemical data supporting the essential role for the B-block histidine and for the absolute sequence conservation of this residue. PMID:19630416
Genome and transcriptome sequencing of lung cancers reveal diverse mutational and splicing events
Liu, Jinfeng; Lee, William; Jiang, Zhaoshi; Chen, Zhongqiang; Jhunjhunwala, Suchit; Haverty, Peter M.; Gnad, Florian; Guan, Yinghui; Gilbert, Houston N.; Stinson, Jeremy; Klijn, Christiaan; Guillory, Joseph; Bhatt, Deepali; Vartanian, Steffan; Walter, Kimberly; Chan, Jocelyn; Holcomb, Thomas; Dijkgraaf, Peter; Johnson, Stephanie; Koeman, Julie; Minna, John D.; Gazdar, Adi F.; Stern, Howard M.; Hoeflich, Klaus P.; Wu, Thomas D.; Settleman, Jeff; de Sauvage, Frederic J.; Gentleman, Robert C.; Neve, Richard M.; Stokoe, David; Modrusan, Zora; Seshagiri, Somasekar; Shames, David S.; Zhang, Zemin
Lung cancer is a highly heterogeneous disease in terms of both underlying genetic lesions and response to therapeutic treatments. We performed deep whole-genome sequencing and transcriptome sequencing on 19 lung cancer cell lines and three lung tumor/normal pairs. Overall, our data show that cell line models exhibit similar mutation spectra to human tumor samples. Smoker and never-smoker cancer samples exhibit distinguishable patterns of mutations. A number of epigenetic regulators, including KDM6A, ASH1L, SMARCA4, and ATAD2, are frequently altered by mutations or copy number changes. A systematic survey of splice-site mutations identified 106 splice site mutations associated with cancer specific aberrant splicing, including mutations in several known cancer-related genes. RAC1b, an isoform of the RAC1 GTPase that includes one additional exon, was found to be preferentially up-regulated in lung cancer. We further show that its expression is significantly associated with sensitivity to a MAP2K (MEK) inhibitor PD-0325901. Taken together, these data present a comprehensive genomic landscape of a large number of lung cancer samples and further demonstrate that cancer-specific alternative splicing is a widespread phenomenon that has potential utility as therapeutic biomarkers. The detailed characterizations of the lung cancer cell lines also provide genomic context to the vast amount of experimental data gathered for these lines over the decades, and represent highly valuable resources for cancer biology. PMID:23033341
RNA-Seq analysis reveals new gene models and alternative splicing in the fungal pathogen Fusarium graminearum
Background The genome of Fusarium graminearum has been sequenced and annotated previously, but correct gene annotation remains a challenge. In addition, posttranscriptional regulations, such as alternative splicing and RNA editing, are poorly understood in F. graminearum. Here we took advantage of RNA-Seq to improve gene annotations and to identify alternative splicing and RNA editing in F. graminearum. Results We identified and revised 655 incorrectly predicted gene models, including revisions of intron predictions, intron splice sites and prediction of novel introns. 231 genes were identified with two or more alternative splice variants, mostly due to intron retention. Interestingly, the expression ratios between different transcript isoforms appeared to be developmentally regulated. Surprisingly, no RNA editing was identified in F. graminearum. Moreover, 2459 novel transcriptionally active regions (nTARs) were identified and our analysis indicates that many of these could be missed genes. Finally, we identified the 5? UTR and/or 3? UTR sequences of 7666 genes. A number of representative novel gene models and alternatively spliced genes were validated by reverse transcription polymerase chain reaction and sequencing of the generated amplicons. Conclusions We have developed novel and efficient strategies to identify alternatively spliced genes and incorrect gene models based on RNA-Seq data. Our study identified hundreds of alternatively spliced genes in F. graminearum and for the first time indicated that alternative splicing is developmentally regulated in filamentous fungi. In addition, hundreds of incorrect predicted gene models were identified and revised and thousands of nTARs were discovered in our study, which will be helpful for the future genomic and transcriptomic studies in F. graminearum. PMID:23324402
Tau Splicing and the Intricacies of Dementia
Andreadis, Athena
Tau is a microtubule associated protein that fulfills several functions critical for neuronal formation and health. Tau discharges its functions by producing multiple isoforms via regulated alternative splicing. These isoforms modulate tau function in normal brain by altering the domains of the protein, thereby influencing its localization, conformation and post-translational modifications and hence its availability and affinity for microtubules and other ligands. Disturbances in tau expression result in disruption of the neuronal cytoskeleton and formation of tau structures (neurofibrillary tangles) found in brains of dementia sufferers. More specifically, aberrations in tau splicing regulation directly cause several neurodegenerative diseases which lead to dementia. In this review, I present our cumulative knowledge of tau splicing regulation in connection with neurodegeneration and also briefly go over the still-extensive list of questions that are connected to tau (dys)function. PMID:21604267
Alternative splicing and gene duplication differentially shaped the regulation of isochorismate synthase in Populus and Arabidopsis
Yuan, Yinan; Chung, Jeng-Der; Fu, Xueyan; Johnson, Virgil E.; Ranjan, Priya; Booth, Sarah L.; Harding, Scott A.; Tsai, Chung-Jui
Isochorismate synthase (ICS) converts chorismate to isochorismate for the biosynthesis of phylloquinone, an essential cofactor for photosynthetic electron transport. ICS is also required for salicylic acid (SA) synthesis during Arabidopsis defense. In several other species, including Populus, SA is derived primarily from the phenylpropanoid pathway. We therefore sought to investigate ICS regulation in Populus to learn the extent of ICS involvement in SA synthesis and defense. Arabidopsis harbors duplicated AtICS genes that differ in their exon-intron structure, basal expression, and stress inducibility. In contrast, we found a single ICS gene in Populus and six other sequenced plant genomes, pointing to the AtICS duplication as a lineage-specific event. The Populus ICS encodes a functional plastidic enzyme, and was not responsive to stresses that stimulated phenylpropanoid accumulation. Populus ICS underwent extensive alternative splicing that was rare for the duplicated AtICSs. Sequencing of 184 RT-PCR Populus clones revealed 37 alternative splice variants, with normal transcripts representing ?50% of the population. When expressed in Arabidopsis, Populus ICS again underwent alternative splicing, but did not produce normal transcripts to complement AtICS1 function. The splice-site sequences of Populus ICS are unusual, suggesting a causal link between junction sequence, alternative splicing, and ICS function. We propose that gene duplication and alternative splicing of ICS evolved independently in Arabidopsis and Populus in accordance with their distinct defense strategies. AtICS1 represents a divergent isoform for inducible SA synthesis during defense. Populus ICS primarily functions in phylloquinone biosynthesis, a process that can be sustained at low ICS transcript levels. PMID:19996170
U2AF1 mutations alter sequence specificity of pre-mRNA binding and splicing.
Okeyo-Owuor, T; White, B S; Chatrikhi, R; Mohan, D R; Kim, S; Griffith, M; Ding, L; Ketkar-Kulkarni, S; Hundal, J; Laird, K M; Kielkopf, C L; Ley, T J; Walter, M J; Graubert, T A
We previously identified missense mutations in the U2AF1 splicing factor affecting codons S34 (S34F and S34Y) or Q157 (Q157R and Q157P) in 11% of the patients with de novo myelodysplastic syndrome (MDS). Although the role of U2AF1 as an accessory factor in the U2 snRNP is well established, it is not yet clear how these mutations affect splicing or contribute to MDS pathophysiology. We analyzed splice junctions in RNA-seq data generated from transfected CD34+ hematopoietic cells and found significant differences in the abundance of known and novel junctions in samples expressing mutant U2AF1 (S34F). For selected transcripts, splicing alterations detected by RNA-seq were confirmed by analysis of primary de novo MDS patient samples. These effects were not due to impaired U2AF1 (S34F) localization as it co-localized normally with U2AF2 within nuclear speckles. We further found evidence in the RNA-seq data for decreased affinity of U2AF1 (S34F) for uridine (relative to cytidine) at the e-3 position immediately upstream of the splice acceptor site and corroborated this finding using affinity-binding assays. These data suggest that the S34F mutation alters U2AF1 function in the context of specific RNA sequences, leading to aberrant alternative splicing of target genes, some of which may be relevant for MDS pathogenesis. PMID:25311244
Maximum mutual information SPLICE transform for seen and unseen conditions
Jasha Droppo; Alex Acero
SPLICE is a front-end technique for automatic speech recog- nition systems. It is a non-linear feature space transformation meant to increase recognition accuracy. Our previous work has shown how to train SPLICE to perform speech feature en- hancement. This paper evaluates a maximum mutual informa- tion (MMI) based discriminative training method for SPLICE. Discriminative techniques tend to excel when the
46 CFR 111.60-19 - Cable splices.
Code of Federal Regulations, 2014 CFR
...Methods � 111.60-19 Cable splices. (a) A cable must not be spliced in a hazardous location, except in intrinsically safe systems. (b) Each cable splice must be made in accordance with section 25.11 of IEEE 45-2002...
46 CFR 111.60-19 - Cable splices.
Code of Federal Regulations, 2012 CFR
...Methods � 111.60-19 Cable splices. (a) A cable must not be spliced in a hazardous location, except in intrinsically safe systems. (b) Each cable splice must be made in accordance with section 25.11 of IEEE 45-2002...
46 CFR 111.60-19 - Cable splices.
Code of Federal Regulations, 2011 CFR
...Methods � 111.60-19 Cable splices. (a) A cable must not be spliced in a hazardous location, except in intrinsically safe systems. (b) Each cable splice must be made in accordance with section 25.11 of IEEE 45-2002...
46 CFR 111.60-19 - Cable splices.
Code of Federal Regulations, 2010 CFR
...Methods � 111.60-19 Cable splices. (a) A cable must not be spliced in a hazardous location, except in intrinsically safe systems. (b) Each cable splice must be made in accordance with section 25.11 of IEEE 45-2002...
46 CFR 111.60-19 - Cable splices.
Code of Federal Regulations, 2013 CFR
...Methods � 111.60-19 Cable splices. (a) A cable must not be spliced in a hazardous location, except in intrinsically safe systems. (b) Each cable splice must be made in accordance with section 25.11 of IEEE 45-2002...
Splicing biomarkers of disease severity in myotonic dystrophy
Nakamori, Masayuki; Sobczak, Krzysztof; Puwanant, Araya; Welle, Steve; Eichinger, Katy; Pandya, Shree; Dekdebrun, Jeannne; Heatwole, Chad R.; McDermott, Michael P.; Chen, Tian; Cline, Melissa; Tawil, Rabi; Osborne, Robert J.; Wheeler, Thurman M.; Swanson, Maurice; Moxley, Richard T.; Thornton, Charles A.
Objective To develop RNA splicing biomarkers of disease severity and therapeutic response in myotonic dystrophy type 1 (DM1) and type 2 (DM2). Methods In a discovery cohort we used microarrays to perform global analysis of alternative splicing in DM1 and DM2. The newly identified splicing changes were combined with previous data to create a panel of 50 putative splicing defects. In a validation cohort of 50 DM1 subjects we measured the strength of ankle dorsiflexion (ADF) and then obtained a needle biopsy of tibialis anterior (TA) to analyze splice events in muscle RNA. The specificity of DM-associated splicing defects was assessed in disease controls. The CTG expansion size in muscle tissue was determined by Southern blot. The reversibility of splicing defects was assessed in transgenic mice by using antisense oligonucleotides (ASOs) to reduce levels of toxic RNA. Results Forty-two splicing defects were confirmed in TA muscle in the validation cohort. Among these, 20 events showed graded changes that correlated with ADF weakness. Five other splice events were strongly affected in DM1 subjects with normal ADF strength. Comparison to disease controls and mouse models indicated that splicing changes were DM-specific, mainly attributable to MBNL1 sequestration, and reversible in mice by targeted knockdown of toxic RNA. Splicing defects and weakness were not correlated with CTG expansion size in muscle tissue. Interpretation Alternative splicing changes in skeletal muscle may serve as biomarkers of disease severity and therapeutic response in myotonic dystrophy. PMID:23929620
Blood Donor Management in China
Shi, Ling; Wang, Jingxing; Liu, Zhong; Stevens, Lori; Sadler, Andrew; Ness, Paul; Shan, Hua
Summary Despite a steady increase in total blood collections and voluntary non-remunerated blood donors, China continues to have many challenges with its blood donation system. The country's donation rate remains low at 9%o, with over 60% of donors being first-time donors. Generally there is a lack of adequate public awareness about blood donation. The conservative donor selection criteria, the relatively long donation interval, and the small donation volume have further limited blood supply. To ensure a sufficient and safe blood supply that meets the increasing clinical need for blood products, there is an urgent need to strengthen the country's blood donor management. This comprehensive effort should include educating and motivating more individuals especially from the rural areas to be involved in blood donation, developing rational and evidence-based selection criteria for donor eligibility, designing a donor follow-up mechanism to encourage more future donations, assessing the current donor testing strategy, improving donor service and care, building regional and national shared donor deferral database, and enhancing the transparency of the blood donation system to gain more trust from the general public. The purpose of the review is to provide an overview of the key process of and challenges with the blood donor management system in China. PMID:25254023
Seth Bates; Tony Smith
\\u000a SharePoint Foundation 2010 provides the core document management, list management, workflow, collaboration, and application\\u000a platform services for a SharePoint environment. SharePoint sites are the foundation on which business solutions based on the\\u000a Office system store and manage information. Sites provide locations where groups of people can work together and share information.\\u000a They can also be used to collect team and
Analysis of Donor Motivations in Living Donor Liver Transplantation
Abdeldayem, Hesham; Kashkoush, Samy; Hegab, Bassem Soliman; Aziz, Amr; Shoreem, Hany; Saleh, Shereef
Objectives: The introduction of the living donor liver transplantation (LDLT) in Egypt as in elsewhere, has raised important psychological conflicts and ethical questions. The objective of this study was to get better understanding of the potential donors� motives toward LDLT. Methods: This study was conducted on consecutive 193 living-liver donors who underwent partial hepatectomy as donors for LDLT during the period between April 2003 and January 2013, at the National Liver Institute Menoufeyia University, Egypt. Potential donors were thoroughly evaluated preoperatively through a screening questionnaire and interviews as regard their demographic data, relationship to the potential recipient, and motives toward proceeding to surgery. They were assured that the information shared between them and the transplant center is confidential. Results: The donors� mean age was 25.53?�?6.39?years with a range of 18�45?years. Males represented 64.7% and females were 35.3%. The most common donors (32.1%, n?=?62) were sons and daughters to their parents (sons: n?=?43, daughters: n?=?19) while parents to their offsprings represent 15% (mothers: n?=?21, fathers: n?=?8). Brothers and sisters represent 16.5% (brothers: n?=?22, sisters: n?=?10). Nephews and nieces giving their uncles or aunts were 14%. The number of wives donating to their husbands was 11 (5.7%). Interestingly, there was no single husband who donated his wife. Among the remaining donors, there were 11 cousins and 1 uncle. Unrelated donors were 20 (10.4%). Several factors seemed to contribute to motivation for donation: the seriousness of the potential recipient condition, the relationship and personal history of the donor to the potential recipient, the religious beliefs, the trust in the health care system, and family dynamics and obligations. Conclusion: Absolute absence of coercion on the living-liver donor�s motives may not be realistic because of the serious condition of the potential recipient. It is mandatory that the donor is truly willing to donate. PMID:25593949
Donor factors influencing graft outcomes in live donor kidney transplantation.
Issa, Naim; Stephany, Brian; Fatica, Richard; Nurko, Saul; Krishnamurthi, Venkatesh; Goldfarb, David A; Braun, William E; Dennis, Vincent W; Heeger, Peter S; Poggio, Emilio D
Living donor renal allograft survival is superior to that achieved from deceased donors, although graft outcome is suboptimal in some of these patients. In an effort to identify the subset of patients at high risk for poor outcomes we studied donor risk factors in 248 living kidney donor-recipient pairs. Unadjusted donor (125)I-iothalamate GFR (iGFR), donor age more than 45 years, donor total cholesterol level less than 200 mg/dL, and donor systolic blood pressure (SBP) less than 120 mm Hg were correlated with allograft estimated glomerular filtration rate (eGFR), and incidence of acute rejection (AR), delayed graft function and/or graft loss at 2 years posttransplantation. Donor iGFR less than 110 mL/min (slope=-7.40, P<0.01), donors more than 45 years (slope=-8.76, P<0.01), donor total cholesterol levels more than 200 mg/dL (slope=-10.03, P<0.01), and SBP more than 120 mm Hg (slope=-5.60, P=0.03) were associated with lower eGFR. By multivariable linear regression analysis these variables remained independently associated with lower eGFR, and poorer outcomes. The increasing number of donor factors (age, iGFR, cholesterol, and blood pressure) was directly associated with worse posttransplant eGFR (P<0.01). In conclusion, our data suggest that routine assessment of living donor parameters could supplement the consideration of recipient characteristics in predicting posttransplant risk of graft injury/dysfunction. PMID:17353780
In vitro self-splicing reactions of the chloroplast group I intron Cr.LSU from Chlamydomonas reinhardtii and in vivo manipulation via gene-replacement.
Thompson, A J; Herrin, D L
The group I intron from the chloroplast rRNA large subunit of Chlamydomonas reinhardtii (Cr.LSU) undergoes autocatalytic splicing in vitro. Cr.LSU displays a range of reactions typical of other group I introns. Under optimal conditions, the 5' cleavage step proceeds rapidly, but the exon-ligation step is relatively slow, and no pH dependent hydrolysis of the 3' splice site occurs. A requirement for high temperature and high [Mg2+] suggests involvement of additional splicing factors in vivo. The positions of three cyclization sites of the free intron have been mapped; two of these sites represent reactions analogous to 5'-splice site cleavage, whereas the third is an example of G-exchange. Cr.LSU contains an open reading frame (ORF) potentially encoding an 163 amino acid polypeptide. ORF function has been investigated by using chloroplast gene replacement via particle bombardment. We have shown that the ORF can be deleted from Cr.LSU without affecting splicing in vivo and it thus does not encode an essential splicing factor. Images PMID:1721704
Differential regulation of alternative 3{prime} splicing of {epsilon} messenger RNA variants
Diaz-Sanchez, D.; Zhang, K.; Saxon, A. [Univ. of California School of Medicine, Los Angeles, CA (United States)] [and others
Alternative 3{prime} splicing of the one active human {epsilon} heavy chain gene results in variants of {epsilon} mRNA encoding distinct IgE proteins. The same relative amounts of these {epsilon} mRNA variants were produced by non-atopic donor B cells when driven in a variety of T-dependent or T-independent systems. The most abundant variants were those for classic secreted {epsilon} and a novel secreted form (CH4-M2{double_prime}). In contrast, cells from subjects with high levels of serum IgE secondary to parasitic infection or atopy spontaneously produced higher relative levels of the CH4-M2{prime} {epsilon} mRNA variant, lower relative amounts of both the membrane and CH4-M2{double_prime} secreted variants, and very low levels of the CH4{prime}-CH5 variant. The existence of and corresponding changes in levels of the CH4-M2{prime}-enclosed secreted protein were demonstrated. IL-10 induced this same differential expression of {epsilon} splice variants in vitro when used to costimulate IL-4 plus CD40-driven B cells and could differentially enhance the production of CH4-M2{prime} protein by established IgE-secreting cell lines. Inhibition of IgE by cross-linking the low affinity IgE receptor (CD23) decreased the levels of {epsilon} mRNA and resulted in a distinct pattern of {epsilon} mRNA characterized by a dramatic decrease in CH4-M2{prime} splice variant. IL-6, IL-2, or IFN-{gamma} did not change the {epsilon} mRNA pattern. Overall, the absolute and relative amounts of the different {epsilon} mRNA splice variants produced appear to be controlled in a differentiation-related fashion.
Comparative analyses between retained introns and constitutively spliced introns in Arabidopsis thaliana using random forest and support vector machine.
Mao, Rui; Raj Kumar, Praveen Kumar; Guo, Cheng; Zhang, Yang; Liang, Chun
One of the important modes of pre-mRNA post-transcriptional modification is alternative splicing. Alternative splicing allows creation of many distinct mature mRNA transcripts from a single gene by utilizing different splice sites. In plants like Arabidopsis thaliana, the most common type of alternative splicing is intron retention. Many studies in the past focus on positional distribution of retained introns (RIs) among different genic regions and their expression regulations, while little systematic classification of RIs from constitutively spliced introns (CSIs) has been conducted using machine learning approaches. We used random forest and support vector machine (SVM) with radial basis kernel function (RBF) to differentiate these two types of introns in Arabidopsis. By comparing coordinates of introns of all annotated mRNAs from TAIR10, we obtained our high-quality experimental data. To distinguish RIs from CSIs, We investigated the unique characteristics of RIs in comparison with CSIs and finally extracted 37 quantitative features: local and global nucleotide sequence features of introns, frequent motifs, the signal strength of splice sites, and the similarity between sequences of introns and their flanking regions. We demonstrated that our proposed feature extraction approach was more accurate in effectively classifying RIs from CSIs in comparison with other four approaches. The optimal penalty parameter C and the RBF kernel parameter [Formula: see text] in SVM were set based on particle swarm optimization algorithm (PSOSVM). Our classification performance showed F-Measure of 80.8% (random forest) and 77.4% (PSOSVM). Not only the basic sequence features and positional distribution characteristics of RIs were obtained, but also putative regulatory motifs in intron splicing were predicted based on our feature extraction approach. Clearly, our study will facilitate a better understanding of underlying mechanisms involved in intron retention. PMID:25110928
Stem�loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly
Sharma, Shalini; Wongpalee, Somsakul Pop; Vashisht, Ajay; Wohlschlegel, James A.; Black, Douglas L.
The pairing of 5? and 3? splice sites across an intron is a critical step in spliceosome formation and its regulation. Interactions that bring the two splice sites together during spliceosome assembly must occur with a high degree of specificity and fidelity to allow expression of functional mRNAs and make particular alternative splicing choices. Here, we report a new interaction between stem�loop 4 (SL4) of the U1 snRNA, which recognizes the 5? splice site, and a component of the U2 small nuclear ribonucleoprotein particle (snRNP) complex, which assembles across the intron at the 3? splice site. Using a U1 snRNP complementation assay, we found that SL4 is essential for splicing in vivo. The addition of free U1-SL4 to a splicing reaction in vitro inhibits splicing and blocks complex assembly prior to formation of the prespliceosomal A complex, indicating a requirement for a SL4 contact in spliceosome assembly. To characterize the interactions of this RNA structure, we used a combination of stable isotope labeling by amino acids in cell culture (SILAC), biotin/Neutravidin affinity pull-down, and mass spectrometry. We show that U1-SL4 interacts with the SF3A1 protein of the U2 snRNP. We found that this interaction between the U1 snRNA and SF3A1 occurs within prespliceosomal complexes assembled on the pre-mRNA. Thus, SL4 of the U1 snRNA is important for splicing, and its interaction with SF3A1 mediates contact between the 5? and 3? splice site complexes within the assembling spliceosome. PMID:25403181
Developmentally regulated alternative splicing of transcripts from the Drosophila homeotic gene Antennapedia can produce four different proteins.
Bermingham, J R; Scott, M P
Antennapedia (Antp) is a Drosophila homeotic gene that controls differentiation of the thoracic segments. Antp transcripts are produced from either of two promoters that are independently regulated in temporally and spatially distinct patterns. In addition, Antp transcripts utilize either of two major polyadenylation sites. Antp primary transcripts contain the same protein coding sequences. Alternative RNA splicing at two positions within the primary transcripts produces mRNAs that can encode four slightly different Antp proteins. Different classes of alternatively spliced transcript predominate early and late in Drosophila development, indicating that the Antp gene is regulated by the processing of its transcripts as well as by controlling their transcription. Alternative splicing appears to be independent of which promoter and which polyadenylation site is used. Images PMID:2903048
A 5' splice-region G----C mutation in exon 1 of the human beta-globin gene inhibits pre-mRNA splicing: a mechanism for beta+-thalassemia.
Vidaud, M; Gattoni, R; Stevenin, J; Vidaud, D; Amselem, S; Chibani, J; Rosa, J; Goossens, M
We have characterized a Mediterranean beta-thalassemia allele containing a sequence change at codon 30 that alters both beta-globin pre-mRNA splicing and the structure of the hemoglobin product. Presumably, this G----C transversion at position -1 of intron 1 reduces severely the utilization of the normal 5' splice site since the level of the Arg----Thr mutant hemoglobin (designated hemoglobin Kairouan) found in the erythrocytes of the patient is very low (2% of total hemoglobin). Since no natural mutations of the guanine located at position -1 of the CAG/GTAAGT consensus sequence had been isolated previously, we investigated the role of this nucleotide in the constitution of an active 5' splice site by studying the splicing of the pre-mRNA in cell-free extracts. We demonstrate that correct splicing of the mutant pre-mRNA is 98% inhibited. Our results provide further insights into the mechanisms of pre-mRNA maturation by revealing that the last residue of the exon plays a role at least equivalent to that of the intron residue at position +5. Images PMID:2915972
SplicePie: a novel analytical approach for the detection of alternative, non-sequential and recursive splicing.
Pulyakhina, Irina; Gazzoli, Isabella; 't Hoen, Peter A C; Verwey, Nisha; den Dunnen, Johan; Aartsma-Rus, Annemieke; Laros, Jeroen F J
Alternative splicing is a powerful mechanism present in eukaryotic cells to obtain a wide range of transcripts and protein isoforms from a relatively small number of genes. The mechanisms regulating (alternative) splicing and the paradigm of consecutive splicing have recently been challenged, especially for genes with a large number of introns. RNA-Seq, a powerful technology using deep sequencing in order to determine transcript structure and expression levels, is usually performed on mature mRNA, therefore not allowing detailed analysis of splicing progression. Sequencing pre-mRNA at different stages of splicing potentially provides insight into mRNA maturation. Although the number of tools that analyze total and cytoplasmic RNA in order to elucidate the transcriptome composition is rapidly growing, there are no tools specifically designed for the analysis of nuclear RNA (which contains mixtures of pre- and mature mRNA). We developed dedicated algorithms to investigate the splicing process. In this paper, we present a new classification of RNA-Seq reads based on three major stages of splicing: pre-, intermediate- and post-splicing. Applying this novel classification we demonstrate the possibility to analyze the order of splicing. Furthermore, we uncover the potential to investigate the multi-step nature of splicing, assessing various types of recursive splicing events. We provide the data that gives biological insight into the order of splicing, show that non-sequential splicing of certain introns is reproducible and coinciding in multiple cell lines. We validated our observations with independent experimental technologies and showed the reliability of our method. The pipeline, named SplicePie, is freely available at: https://github.com/pulyakhina/splicing_analysis_pipeline. The example data can be found at: https://barmsijs.lumc.nl/HG/irina/example_data.tar.gz. PMID:25800735
UnSplicer: mapping spliced RNA-Seq reads in compact genomes and filtering noisy splicing.
Burns, Paul D; Li, Yang; Ma, Jian; Borodovsky, Mark
Accurate mapping of spliced RNA-Seq reads to genomic DNA has been known as a challenging problem. Despite significant efforts invested in developing efficient algorithms, with the human genome as a primary focus, the best solution is still not known. A recently introduced tool, TrueSight, has demonstrated better performance compared with earlier developed algorithms such as TopHat and MapSplice. To improve detection of splice junctions, TrueSight uses information on statistical patterns of nucleotide ordering in intronic and exonic DNA. This line of research led to yet another new algorithm, UnSplicer, designed for eukaryotic species with compact genomes where functional alternative splicing is likely to be dominated by splicing noise. Genome-specific parameters of the new algorithm are generated by GeneMark-ES, an ab initio gene prediction algorithm based on unsupervised training. UnSplicer shares several components with TrueSight; the difference lies in the training strategy and the classification algorithm. We tested UnSplicer on RNA-Seq data sets of Arabidopsis thaliana, Caenorhabditis elegans, Cryptococcus neoformans and Drosophila melanogaster. We have shown that splice junctions inferred by UnSplicer are in better agreement with knowledge accumulated on these well-studied genomes than predictions made by earlier developed tools. PMID:24259430
Development of therapeutic splice-switching oligonucleotides.
Disterer, Petra; Kryczka, Adrianna; Liu, Yuqi; Badi, Yusef E; Wong, Jessie J; Owen, James S; Khoo, Bernard
Synthetic splice-switching oligonucleotides (SSOs) target nuclear pre-mRNA molecules to change exon splicing and generate an alternative protein isoform. Clinical trials with two competitive SSO drugs are underway to treat Duchenne muscular dystrophy (DMD). Beyond DMD, many additional therapeutic applications are possible, with some in phase 1 clinical trials or advanced preclinical evaluation. Here, we present an overview of the central factors involved in developing therapeutic SSOs for the treatment of diseases. The selection of susceptible pre-mRNA target sequences, as well as the design and chemical modification of SSOs to increase SSO stability and effectiveness, are key initial considerations. Identification of effective SSO target sequences is still largely empirical and published guidelines are not a universal guarantee for success. Specifically, exon-targeted SSOs, which are successful in modifying dystrophin splicing, can be ineffective for splice-switching in other contexts. Chemical modifications, importantly, are associated with certain characteristic toxicities, which need to be addressed as target diseases require chronic treatment with SSOs. Moreover, SSO delivery in adequate quantities to the nucleus of target cells without toxicity can prove difficult. Last, the means by which these SSOs are administered needs to be acceptable to the patient. Engineering an efficient therapeutic SSO, therefore, necessarily entails a compromise between desirable qualities and effectiveness. Here, we describe how the application of optimal solutions may differ from case to case. PMID:24826963
Detect Digital Image Splicing with Visual Cues
Aickelin, Uwe
present experimental results which show that the proposed system outperforms the prior arts. An additional techniques to create forgery images. As shown in Fig. 1, by copying a spliced portion from the source image into a target image, one can create a composite scenery to cheat others. Helped by current state-of-the-art
DNA Computing Based on Splicing: Universality Results
Kari, Lila
/absence of certain symbols in the spliced strings, and organizing the work of the system in a distributed way (like, hence the aim is to reach the power of Chomsky type-0 grammars, the standard model for representing was the announcement of solving (a small instance of) the Hamiltonian path problem in a test tube just by handling DNA
The genetics of splicing in neuroblastoma
Regulation of mRNA splicing, a critical and tightly regulated cellular function, underlies the majority of proteomic diversity, and is frequently disrupted in disease. Using an integrative genomics approach, we combined both genome and exon level transcriptome data in two somatic tissues (cerebella and peripheral ganglia) from a transgenic mouse model of neuroblastoma, a tumor that arises from peripheral neural crest.
Recognition of Unknown Conserved Alternatively Spliced Exons
Uwe Ohler; Noam Shomron; Christopher B. Burge
The split structure of most mammalian protein-coding genes allows for the potential to produce multiple different mRNA and protein isoforms from a single gene locus through the process of alternative splicing (AS). We propose a computational approach called UNCOVER based on a pair hidden Markov model to discover conserved coding exonic sequences subject to AS that have so far gone
Alternative splicing of DNA damage response genes and gastrointestinal cancers
Rahmutulla, Bahityar; Matsushita, Kazuyuki; Nomura, Fumio
Alternative splicing, which is a common phenomenon in mammalian genomes, is a fundamental process of gene regulation and contributes to great protein diversity. Alternative splicing events not only occur in the normal gene regulation process but are also closely related to certain diseases including cancer. In this review, we briefly demonstrate the concept of alternative splicing and DNA damage and describe the association of alternative splicing and cancer pathogenesis, focusing on the potential relationship of alternative splicing, DNA damage, and gastrointestinal cancers. We will also discuss whether alternative splicing leads to genetic instability, which is considered to be a driving force for tumorigenesis. Better understanding of the role and mechanism of alternative splicing in tumorigenesis may provide new directions for future cancer studies. PMID:25516641
The genetic basis for individual differences in mRNA splicing and APOBEC1 editing activity in murine macrophages
Hassan, Musa A.; Butty, Vincent; Jensen, Kirk D.C.; Saeij, Jeroen P.J.
Alternative splicing and mRNA editing are known to contribute to transcriptome diversity. Although alternative splicing is pervasive and contributes to a variety of pathologies, including cancer, the genetic context for individual differences in isoform usage is still evolving. Similarly, although mRNA editing is ubiquitous and associated with important biological processes such as intracellular viral replication and cancer development, individual variations in mRNA editing and the genetic transmissibility of mRNA editing are equivocal. Here, we have used linkage analysis to show that both mRNA editing and alternative splicing are regulated by the macrophage genetic background and environmental cues. We show that distinct loci, potentially harboring variable splice factors, regulate the splicing of multiple transcripts. Additionally, we show that individual genetic variability at the Apobec1 locus results in differential rates of C-to-U(T) editing in murine macrophages; with mouse strains expressing mostly a truncated alternative transcript isoform of Apobec1 exhibiting lower rates of editing. As a proof of concept, we have used linkage analysis to identify 36 high-confidence novel edited sites. These results provide a novel and complementary method that can be used to identify C-to-U editing sites in individuals segregating at specific loci and show that, beyond DNA sequence and structural changes, differential isoform usage and mRNA editing can contribute to intra-species genomic and phenotypic diversity. PMID:24249727
Donor compensation: an ethical imperative!
Reichardt, J-O
The number of living organ donors is increasing worldwide, but donor needs are widely neglected in support of anticommodification policies. This article argues that the warrant of donor autonomy during the decision process to donate is only one requirement of adequate donor care. Another is the donor's protection against the systematic and institutional exploitation of his altruistic dispositions. People with the disposition to support those, who are in desperate need, with a nonrenewable part of their own body, despite a small but unavoidable risk of death or health impairment, do not deserve to be additionally burdened with further disincentives, such as financial risks and uncompensated costs of donation. And although the borderline between a morally required disincentive removal and a more controversial net incentive to boost donation might be vague and open to discussion, to disadvantage living donors by design constitutes a serious barrier to the fairness of living organ donation-a barrier that should be removed. PMID:20172295
A mutation in a rare type of intron in a sodium-channel gene results in aberrant splicing and causes myotonia
Kubota, Tomoya; Roca, Xavier; Kimura, Takashi; Kokunai, Yosuke; Nishino, Ichizo; Sakoda, Saburo; Krainer, Adrian R.; Takahashi, Masanori P.
Many mutations in the skeletal-muscle sodium-channel gene SCN4A have been associated with myotonia and/or periodic paralysis, but so far all of these mutations are located in exons. We found a patient with myotonia caused by a deletion/insertion located in intron 21 of SCN4A, which is an AT-AC type II intron. This is a rare class of introns that, despite having AT-AC boundaries, are spliced by the major or U2-type spliceosome. The patient's skeletal muscle expressed aberrantly spliced SCN4A mRNA isoforms generated by activation of cryptic splice sites. In addition, genetic suppression experiments using an SCN4A minigene showed that the mutant 5? splice site has impaired binding to the U1 and U6 snRNPs, which are the cognate factors for recognition of U2-type 5? splice sites. One of the aberrantly spliced isoforms encodes a channel with a 35-amino-acid insertion in the cytoplasmic loop between domains III and IV of Nav1.4. The mutant channel exhibited a marked disruption of fast inactivation, and a simulation in silico showed that the channel defect is consistent with the patient's myotonic symptoms. This is the first report of a disease-associated mutation in an AT-AC type II intron, and also the first intronic mutation in a voltage-gated ion channel gene showing a gain-of-function defect. PMID:21412952
CD20 alternative splicing isoform generates immunogenic CD4 helper T epitopes.
Vauchy, Charline; Gamonet, Clementine; Ferrand, Christophe; Daguindau, Etienne; Galaine, Jeanne; Beziaud, Laurent; Chauchet, Adrien; Henry Dunand, Carole J; Deschamps, Marina; Rohrlich, Pierre Simon; Borg, Christophe; Adotevi, Olivier; Godet, Yann
Cancer-specific splice variants gain significant interest as they generate neo-antigens that could be targeted by immune cells. CD20, a membrane antigen broadly expressed in mature B cells and in B cell lymphomas, is subject to an alternative splicing named D393-CD20 leading to loss of membrane expression of the spliced isoform. D393-CD20 expression is detectable in transformed B cells and upregulated in various lymphoma B cells. In this study, we show that D393-CD20 is translated in malignant B cells and that D393-CD20 specific CD4 T cells producing IFN-? are present in B-cell lymphoma patients. Then, we have investigated whether the 20mer D393-CD20 peptide spanning the splicing site might be targeted by the immune system and we have shown that D393-CD20-specific CD4 Th1 clones could directly recognize malignant B cell lines and kill autologous lymphoma B cells indicating that D393-CD20-derived epitopes are naturally processed and presented on tumor cells. Finally, D393-CD20 peptide-based vaccination induced specific CD8 and CD4 T cell responses in HLA-humanized transgenic mice suggesting the presentation of D393-CD20 derived peptides on both HLA Class-I and -II. These findings support further investigations on the potential use of D393-CD20 directed specific immunotherapy in B cell malignancies. PMID:25449106
A serine�arginine-rich (SR) splicing factor modulates alternative splicing of over a thousand genes in Toxoplasma gondii
Yeoh, Lee M.; Goodman, Christopher D.; Hall, Nathan E.; van�Dooren, Giel G.; McFadden, Geoffrey I.; Ralph, Stuart A.
Single genes are often subject to alternative splicing, which generates alternative mature mRNAs. This phenomenon is widespread in animals, and observed in over 90% of human genes. Recent data suggest it may also be common in Apicomplexa. These parasites have small genomes, and economy of DNA is evolutionarily favoured in this phylum. We investigated the mechanism of alternative splicing in Toxoplasma gondii, and have identified and localized TgSR3, a homologue of ASF/SF2 (alternative-splicing factor/splicing factor 2, a serine-arginine�rich, or SR protein) to a subnuclear compartment. In addition, we conditionally overexpressed this protein, which was deleterious to growth. qRT-PCR was used to confirm perturbation of splicing in a known alternatively-spliced gene. We performed high-throughput RNA-seq to determine the extent of splicing modulated by this protein. Current RNA-seq algorithms are poorly suited to compact parasite genomes, and hence we complemented existing tools by writing a new program, GeneGuillotine, that addresses this deficiency by segregating overlapping reads into distinct genes. In order to identify the extent of alternative splicing, we released another program, JunctionJuror, that detects changes in intron junctions. Using this program, we identified about 2000 genes that were constitutively alternatively spliced in T. gondii. Overexpressing the splice regulator TgSR3 perturbed alternative splicing in over 1000 genes. PMID:25870410
SpliceMiner: a high-throughput database implementation of the NCBI Evidence Viewer for microarray splice variant analysis
Kahn, Ari B; Ryan, Michael C; Liu, Hongfang; Zeeberg, Barry R; Jamison, D Curtis; Weinstein, John N
Background There are many fewer genes in the human genome than there are expressed transcripts. Alternative splicing is the reason. Alternatively spliced transcripts are often specific to tissue type, developmental stage, environmental condition, or disease state. Accurate analysis of microarray expression data and design of new arrays for alternative splicing require assessment of probes at the sequence and exon levels. Description SpliceMiner is a web interface for querying Evidence Viewer Database (EVDB). EVDB is a comprehensive, non-redundant compendium of splice variant data for human genes. We constructed EVDB as a queryable implementation of the NCBI Evidence Viewer (EV). EVDB is based on data obtained from NCBI Entrez Gene and EV. The automated EVDB build process uses only complete coding sequences, which may or may not include partial or complete 5' and 3' UTRs, and filters redundant splice variants. Unlike EV, which supports only one-at-a-time queries, SpliceMiner supports high-throughput batch queries and provides results in an easily parsable format. SpliceMiner maps probes to splice variants, effectively delineating the variants identified by a probe. Conclusion EVDB can be queried by gene symbol, genomic coordinates, or probe sequence via a user-friendly web-based tool we call SpliceMiner (). The EVDB/SpliceMiner combination provides an interface with human splice variant information and, going beyond the very valuable NCBI Evidence Viewer, supports fluent, high-throughput analysis. Integration of EVDB information into microarray analysis and design pipelines has the potential to improve the analysis and bioinformatic interpretation of gene expression data, for both batch and interactive processing. For example, whenever a gene expression value is recognized as important or appears anomalous in a microarray experiment, the interactive mode of SpliceMiner can be used quickly and easily to check for possible splice variant issues. PMID:17338820
Missense mutations in cancer suppressor gene TP53 are colocalized with exonic splicing enhancers (ESEs).
Gorlov, Ivan P; Gorlova, Olga Y; Frazier, Marsha L; Amos, Christopher I
Mutation databases can be viewed as footprints of functional organization of a gene and thus can be used to infer its functional organization. We studied the association of exonic splicing enhancers (ESEs) with missense mutations in the tumor suppressor gene TP53 using the International Agency for Research on Cancer (IARC) mutation database. The goals of the study were: (i) to verify the hypothesis that deleterious missense mutations are colocalized with ESEs; (ii) to identify potentially functional ESE sites in the open reading frame (ORF) of the TP53. If some sequence functions as a splicing enhancer, then nucleotide substitutions in the site will disturb splicing, abrogate p53 function, and cause an increased susceptibility to cancer. Therefore, among cancers showing p53 mutations, more missense mutations are expected within functional ESE sites as compared to non-functional ESE motifs. Using several statistical tests, we found that missense mutations in TP53 are strongly colocalized with ESEs, and that only a small fraction of ESE sites contributes to the association. There are usually one or two ESEs per exon showing a statistically significant association with missense mutations--so-called significant ESE sites. In many respects significant ESE sites are different from those that do not show association with missense mutations. We found that positions of significant ESE sites are codon-dependent--significant ESEs preferentially start from the first position of a codon, whereas non-significant ESEs show no position dependence. Significant ESEs showed a more limited set of sequences compared to non-significant ESEs. These findings suggest that there is a limited number of missense mutations that influence ESE sites and our analysis provides further insight into the types of sites that harbor exonic enhancer elements. PMID:15450416
Late Infantile Neuronal Ceroid Lipofuscinosis Is Due to Splicing Mutations in the CLN2 Gene
Jaana M. Hartikainen; Weina Ju; Krystyna E. Wisniewski; Dorota N. Moroziewicz; Alexandra L. Kaczmarski; Lucille McLendon; David Zhong; Carolin T. Suarez; W. Ted Brown; Nan Zhong
Late infantile neuronal ceroid lipofuscinosis, LINCL, is one of the most common pediatric neurodegenerative disorders. It is caused by mutations in the CLN2 gene, which encodes a lysosomal pepstatin-insensitive peptidase (LPIP). We have identified a novel mutation, T523-1G ? A, by molecular analyses of three unrelated LINCL cases. The mutation was found to affect a 3? intronic splicing acceptor site,
Structure of Slitrk2-PTP? complex reveals mechanisms for splicing-dependent trans-synaptic adhesion.
Yamagata, Atsushi; Sato, Yusuke; Goto-Ito, Sakurako; Uemura, Takeshi; Maeda, Asami; Shiroshima, Tomoko; Yoshida, Tomoyuki; Fukai, Shuya
Selective binding between pre- and postsynaptic adhesion molecules can induce synaptic differentiation. Here we report the crystal structure of a synaptogenic trans-synaptic adhesion complex between Slit and Trk-like family member 2 (Slitrk2) and receptor protein tyrosine phosphatase (RPTP) ?. The structure and site-directed mutational analysis revealed the structural basis of splicing-dependent adhesion between Slitrks and type IIa RPTPs for inducing synaptic differentiation. PMID:25989451
Structure of Slitrk2�PTP? complex reveals mechanisms for splicing-dependent trans-synaptic adhesion
Yamagata, Atsushi; Sato, Yusuke; Goto-Ito, Sakurako; Uemura, Takeshi; Maeda, Asami; Shiroshima, Tomoko; Yoshida, Tomoyuki; Fukai, Shuya
Selective binding between pre- and postsynaptic adhesion molecules can induce synaptic differentiation. Here we report the crystal structure of a synaptogenic trans-synaptic adhesion complex between Slit and Trk-like family member 2 (Slitrk2) and receptor protein tyrosine phosphatase (RPTP) ?. The structure and site-directed mutational analysis revealed the structural basis of splicing-dependent adhesion between Slitrks and type IIa RPTPs for inducing synaptic differentiation. PMID:25989451
The ASAP II database: analysis and comparative genomics of alternative splicing in 15 animal species
Namshin Kim; Alexander V. Alekseyenko; Meenakshi Roy; Christopher Lee
We have greatly expanded the Alternative Splicing Annotation Project (ASAP) database: (i) its human alternative splicing data are expanded ? 3-fold over the previous ASAP database, to nearly 90000 distinct alternative splicing events; (ii) it now provides genome-wide alternative splicing analyses for 15 vertebrate, insect and other animal species; (iii) it provides comprehensive comparative genomics information for comparing alternative splicing
The influence of Argonaute proteins on alternative RNA splicing.
Batsch�, Eric; Ameyar-Zazoua, Maya
Alternative splicing of precursor RNAs is an important process in multicellular species because it impacts several aspects of gene expression: from the increase of protein repertoire to the level of expression. A large body of evidences demonstrates that factors regulating chromatin and transcription impact the outcomes of alternative splicing. Argonaute (AGO) proteins were known to play key roles in the regulation of gene expression at the post-transcriptional level. More recently, their role in the nucleus of human somatic cells has emerged. Here, we will discuss some of the nuclear functions of AGO, with special emphasis on alternative splicing. The AGO-mediated modulation of alternative splicing is based on several properties of these proteins: their binding to transcripts on chromatin and their interactions with many proteins, especially histone tail-modifying enzymes, HP1? and splicing factors. AGO proteins may favor a decrease in the RNA-polymerase II kinetics at actively transcribed genes leading to the modulation of alternative splicing decisions. They could also influence alternative splicing through their interaction with core components of the splicing machinery and several splicing factors. We will discuss the modes of AGO recruitment on chromatin at active genes. We suggest that long intragenic antisense transcripts (lincRNA) might be an important feature of genes containing splicing events regulated by AGO. PMID:25255778
Marrow transplantation from unrelated donors.
Sierra, J; Anasetti, C
The use of an HLA-compatible unrelated donor is an option for patients who require an allogeneic transplant but lack a family member match. Grafts from unrelated volunteer donors have provided long-term disease-free survival for a variable proportion of patients, depending on degree of HLA matching with the donor, patient's disease, disease stage, and age. The number of volunteers in marrow donor registries worldwide has increased to more than 2.5 million. The number of unrelated donor transplants facilitated by the US National Marrow Donor Program alone will exceed 900 this year. Progress in HLA-typing technology results in a more precise definition of donor and recipient matching and new assays have been developed with initial success to measure alloreactive T-cell precursors for selection of donors with less antihost reactivity. Prevention and treatment of graft failure, graft-versus-host disease, opportunistic infections, and Epstein-Barr virus-associated lymphoproliferative disease remain a challenge. PMID:9372034
Donor Preferences and Charitable Giving
ERIC Educational Resources Information Center
Williams, Stephanie Roderick
This study aimed to learn more of the differences that may exist between the two most powerful groups of donors today, baby boomers (40-58 years old) and mature donors (59 and older), in an effort to help organizations improve fundraising efforts. Questions about the importance of organizational efficiency, program outcomes, and the desire for�
ERIC Educational Resources Information Center
Walker, Mary Margaret
A discussion of donor records in college and university fund-raising programs looks at a variety of issues, including who sees them (administrators, donors, volunteers, and members of the legal profession), how access to them is controlled, and what is kept in them. Suggestions are offered for managing such records, and the experiences of a number�
Donor registries and search strategies.
Hurley, Carolyn K; Oudshoorn, Machteld; Setterholm, Michelle
The optimal donor of hematopoietic progenitor cells shares alleles of the major histocompatibility genes with the recipient. This chapter describes the strategies aimed at identifying such a matched donor from registries of volunteers or from umbilical cord blood banks. PMID:22665254
Polypyrimidine tract binding protein inhibits IgM pre-mRNA splicing by diverting U2 snRNA base-pairing away from the branch point.
Zheng, Xuexiu; Cho, Sunghee; Moon, Heegyum; Loh, Tiing Jen; Oh, Huyn Kyung; Green, Michael R; Shen, Haihong
The mouse immunoglobulin (IgM) pre-mRNA contains a splicing inhibitor that bears multiple binding sites for the splicing repressor polypyrimidine tract binding protein (PTB). Here we show that the inhibitor directs assembly of an ATP-dependent complex that contains PTB and U1 and U2 small nuclear RNAs (snRNAs). Unexpectedly, although U2 snRNA is present in the inhibitor complex, it is not base-paired to the branch point. We present evidence that inhibitor-bound PTB contacts U2 snRNA to promote base-pairing to an adjacent branch point-like sequence within the inhibitor, thereby preventing the U2 snRNA-branch point interaction and resulting in splicing repression. Our studies reveal a novel mechanism by which PTB represses splicing. PMID:24572809
hnRNP U protein is required for normal pre-mRNA splicing and postnatal heart development and function.
Ye, Junqiang; Beetz, Nadine; O'Keeffe, Sean; Tapia, Juan Carlos; Macpherson, Lindsey; Chen, Weisheng V; Bassel-Duby, Rhonda; Olson, Eric N; Maniatis, Tom
We report that mice lacking the heterogeneous nuclear ribonucleoprotein U (hnRNP U) in the heart develop lethal dilated cardiomyopathy and display numerous defects in cardiac pre-mRNA splicing. Mutant hearts have disorganized cardiomyocytes, impaired contractility, and abnormal excitation-contraction coupling activities. RNA-seq analyses of Hnrnpu mutant hearts revealed extensive defects in alternative splicing of pre-mRNAs encoding proteins known to be critical for normal heart development and function, including Titin and calcium/calmodulin-dependent protein kinase II delta (Camk2d). Loss of hnRNP U expression in cardiomyocytes also leads to aberrant splicing of the pre-mRNA encoding the excitation-contraction coupling component Junctin. We found that the protein product of an alternatively spliced Junctin isoform is N-glycosylated at a specific asparagine site that is required for interactions with specific protein partners. Our findings provide conclusive evidence for the essential role of hnRNP U in heart development and function and in the regulation of alternative splicing. PMID:26039991
Visualizing group II intron catalysis through the stages of splicing
Marcia, Marco; Pyle, Anna Marie
SUMMARY Group II introns are self-splicing ribozymes that share a reaction mechanism and a common ancestor with the eukaryotic spliceosome, thereby providing a model system for understanding the chemistry of pre-mRNA splicing. Here we report fourteen crystal structures of a group II intron at different stages of catalysis. We provide a detailed mechanism for the first step of splicing, we describe a reversible conformational change between the first and the second steps of splicing, and we present the ligand-free intron structure after splicing, in an active state that corresponds to the retrotransposable form of the intron. During each reaction, the reactants are aligned and activated by a heteronuclear four-metal-ion center that contains a metal cluster and obligate monovalent cations, adopting a structural arrangement similar to that of protein endonucleases. Based on our data, we propose a model for the splicing cycle and show that it is applicable to the eukaryotic spliceosome. PMID:23101623
Dysfunctional Gene Splicing as a Potential Contributor to Neuropsychiatric Disorders
Glatt, Stephen J.; Cohen, Ori S.; Faraone, Stephen V.; Tsuang, Ming T.
Alternative pre-mRNA splicing is a major mechanism by which the proteomic diversity of eukaryotic genomes is amplified. Much akin to neuropsychiatric disorders themselves, alternative splicing events can be influenced by genetic, developmental, and environmental factors. Here we review the evidence that abnormalities of splicing may contribute to the liability toward these disorders. First, we introduce the phenomenon of alternative splicing and describe the processes involved in its regulation. We then review the evidence for specific splicing abnormalities in a wide range of neuropsychiatric disorders, including psychotic disorders (schizophrenia), affective disorders (bipolar disorder and major depressive disorder), suicide, substance abuse disorders (cocaine abuse and alcoholism), and neurodevelopmental disorders (autism). Next, we provide a theoretical reworking of the concept of �gene-focused� epidemiologic and neurobiologic investigations. Lastly, we suggest potentially fruitful lines for future research that should illuminate the nature, extent, causes, and consequences of alternative splicing abnormalities in neuropsychiatric disorders. PMID:21438146
A CTD function linking transcription to splicing
Jeffry L. Corden; Meera Patturajan
Since its discovery in 1985, the function of the C-terminal domain (CTD) of RNA polymerase li has been a puzzle. Recent studies suggest that the CTD functions as a linear platform for assembly of complexes that splice, cleave and polyadenylate pre-mRNA. A new set of CTD-associated SR-like proteins (CASPs) have been implicated in pre-mRNA processing and transcription elongation as a
Splicing variants of porcine synphilin-1?
Larsen, Knud; Madsen, Lone Bruhn; Farajzadeh, Leila; Bendixen, Christian
Parkinson's disease (PD), idiopathic and familial, is characterized by degradation of dopaminergic neurons and the presence of Lewy bodies (LB) in the substantia nigra. LBs contain aggregated proteins of which ?-synuclein is the major component. The protein synphilin-1 interacts and colocalizes with ?-synuclein in LBs. The aim of this study was to isolate and characterize porcine synphilin-1 and isoforms hereof with the future perspective to use the pig as a model for Parkinson's disease. The porcine SNCAIP cDNA was cloned by reverse transcriptase PCR. The spatial expression of SNCAIP mRNA was investigated by RNAseq. The presented work reports the molecular cloning and characterization of the porcine (Sus scrofa) synphilin-1 cDNA (SNCAIP) and three splice variants hereof. The porcine SNCAIP cDNA codes for a protein (synphilin-1) of 919 amino acids which shows a high similarity to human (90%) and to mouse (84%) synphilin-1. Three shorter transcript variants of the synphilin-1 gene were identified, all lacking one or more exons. SNCAIP transcripts were detected in most examined organs and tissues and the highest expression was found in brain tissues and lung. Conserved splicing variants and a novel splice form of synhilin-1 were found in this study. All synphilin-1 isoforms encoded by the identified transcript variants lack functional domains important for protein degradation.
Splicing variants of porcine synphilin-1.
Larsen, Knud; Madsen, Lone Bruhn; Farajzadeh, Leila; Bendixen, Christian
Parkinson's disease (PD), idiopathic and familial, is characterized by degradation of dopaminergic neurons and the presence of Lewy bodies (LB) in the substantia nigra. LBs contain aggregated proteins of which ?-synuclein is the major component. The protein synphilin-1 interacts and colocalizes with ?-synuclein in LBs. The aim of this study was to isolate and characterize porcine synphilin-1 and isoforms hereof with the future perspective to use the pig as a model for Parkinson's disease. The porcine SNCAIP cDNA was cloned by reverse transcriptase PCR. The spatial expression of SNCAIP mRNA was investigated by RNAseq. The presented work reports the molecular cloning and characterization of the porcine (Sus scrofa) synphilin-1 cDNA (SNCAIP) and three splice variants hereof. The porcine SNCAIP cDNA codes for a protein (synphilin-1) of 919 amino acids which shows a high similarity to human (90%) and to mouse (84%) synphilin-1. Three shorter transcript variants of the synphilin-1 gene were identified, all lacking one or more exons. SNCAIP transcripts were detected in most examined organs and tissues and the highest expression was found in brain tissues and lung. Conserved splicing variants and a novel splice form of synhilin-1 were found in this study. All synphilin-1 isoforms encoded by the identified transcript variants lack functional domains important for protein degradation. PMID:26101749
Alternative splicing is frequent during early embryonic development in mouse
Timoth�e Revil; Daniel Gaffney; Christel Dias; Jacek Majewski; Loydie A Jerome-Majewska
BACKGROUND: Alternative splicing is known to increase the complexity of mammalian transcriptomes since nearly all mammalian genes express multiple pre-mRNA isoforms. However, our knowledge of the extent and function of alternative splicing in early embryonic development is based mainly on a few isolated examples. High throughput technologies now allow us to study genome-wide alternative splicing during mouse development. RESULTS: A
Neuronal Signaling through Alternative Splicing: Some Exons CaRRE...
NSDL National Science Digital Library
Kevin J. O'Donovan (The Rockefeller University; Laboratory of Molecular Neuro-Oncology REV)
Alternative splicing represents a mechanism by which a single gene can be used to create proteins with different functions. Neurons use alternative splicing to produce channels with different sequences and biophysical or regulatory properties. O'Donovan and Darnell discuss a mechanism by which neurons can alter channel splicing in response to neuronal activity through a signal generated by calcium and calcium/calmodulin-dependent kinase activity.
The Caenorhabditis elegans Gene mfap-1 Encodes a Nuclear Protein That Affects Alternative Splicing
Horvitz, H Robert
RNA splicing is a major regulatory mechanism for controlling eukaryotic gene expression. By generating various splice isoforms from a single pre�mRNA, alternative splicing plays a key role in promoting the evolving complexity ...
PMD patient mutations reveal a long-distance intronic interaction that regulates PLP1/DM20 alternative splicing.
Taube, Jennifer R; Sperle, Karen; Banser, Linda; Seeman, Pavel; Cavan, Barbra Charina V; Garbern, James Y; Hobson, Grace M
Alternative splicing of the proteolipid protein 1 gene (PLP1) produces two forms, PLP1 and DM20, due to alternative use of 5' splice sites with the same acceptor site in intron 3. The PLP1 form predominates in central nervous system RNA. Mutations that reduce the ratio of PLP1 to DM20, whether mutant or normal protein is formed, result in the X-linked leukodystrophy Pelizaeus-Merzbacher disease (PMD). We investigated the ability of sequences throughout PLP1 intron 3 to regulate alternative splicing using a splicing minigene construct transfected into the oligodendrocyte cell line, Oli-neu. Our data reveal that the alternative splice of PLP1 is regulated by a long-distance interaction between two highly conserved elements that are separated by 581 bases within the 1071-base intron 3. Further, our data suggest that a base-pairing secondary structure forms between these two elements, and we demonstrate that mutations of either element designed to destabilize the secondary structure decreased the PLP1/DM20 ratio, while swap mutations designed to restore the structure brought the PLP1/DM20 ratio to near normal levels. Sequence analysis of intron 3 in families with clinical symptoms of PMD who did not have coding-region mutations revealed mutations that segregated with disease in three families. We showed that these patient mutations, which potentially destabilize the secondary structure, also reduced the PLP1/DM20 ratio. This is the first report of patient mutations causing disease by disruption of a long-distance intronic interaction controlling alternative splicing. This finding has important implications for molecular diagnostics of PMD. PMID:24890387
Living Kidney Donors and ESRD.
Ross, Lainie Friedman
There are more than 325 living kidney donors who have developed end-stage renal disease and have been listed on the Organ Procurement and Transplantation Network (OPTN)/United Network for Organ Sharing (UNOS) deceased donor kidney wait list. The OPTN/UNOS database records where these kidney donors are listed and, if they donated after April 1994, where that donation occurred. These 2 locations are often not the same. In this commentary, I examine whether a national living donor registry should be created and whether transplantation centers should be notified when one of their living kidney donors develops end-stage renal disease. I consider and refute 5 potential objections to center notification. I explain that transplantation centers should look back at these cases and input data into a registry to attempt to identify patterns that could improve donor evaluation protocols. Creating a registry and mining the information it contains is, in my view, our moral and professional responsibility to future patients and the transplantation endeavor. As individuals and as a community, we need to acknowledge the many unknown risks of living kidney donation and take responsibility for identifying these risks. We then must share information about these risks, educate prospective donors about them, and attempt to minimize them. PMID:25936672
Live Donors in Liver Transplantation
Brown, Robert S.
Living donor liver transplantation (LDLT) has been controversial since its inception. Begun in response to deceased donor organ shortage and waiting list mortality, LDLT was initiated in 1989 in children, grew rapidly after its first general application in adults in the US in 1998, and has declined since 2001. There are significant risks to the living donor, including the risk of death and substantial morbidity, and two highly publicized donor deaths are thought to have contributed to decreased enthusiasm for LDLT. Significant improvements in outcomes have been seen over recent years and data, including from the NIH-funded Adult-to-Adult Living Donor Liver Transplantation Cohort Study, A2ALL, has established a survival benefit from pursuing LDLT. Despite this, LDLT still comprises less than 5% of adult liver transplants, significantly less than in kidney transplantation where living donors comprise approximately 40% of all transplant performed. The ethics, optimal utility and application of LDLT remain to be defined. In addition, most studies to date have focused on post-transplant outcomes and not included the effect of the learning curve on outcome or the potential impact of LDLT on waiting list mortality. Further growth of LDLT will depend on defining the optimal recipient and donor characteristics for this procedure as well as broader acceptance and experience in the public and in transplant centers. PMID:18471556
Disturbed Expression of Splicing Factors in Renal Cancer Affects Alternative Splicing of Apoptosis Regulators, Oncogenes, and Tumor Suppressors
Agnieszka Piekielko-Witkowska; Hanna Wiszomirska; Anna Wojcicka; Piotr Poplawski; Joanna Boguslawska; Zbigniew Tanski; Alicja Nauman; Juan Valcarcel
BackgroundClear cell renal cell carcinoma (ccRCC) is the most common type of renal cancer. One of the processes disturbed in this cancer type is alternative splicing, although phenomena underlying these disturbances remain unknown. Alternative splicing consists of selective removal of introns and joining of residual exons of the primary transcript, to produce mRNA molecules of different sequence. Splicing aberrations may
To determine possible causes of declining Rio Grande wild turkey (RGWT; Meleagris gallopavo intermedia) abundance in the southern Edwards Plateau, research was conducted on 4 sites, 2 with stable (S [SA and SB]; Kerr and Real counties) and 2...
Identification of the alternative splicing of the UL49 locus of human cytomegalovirus.
Yang, Guang; Li, Wei; Liao, Wenzhen; Zhang, Xin; Zou, Yi; Dai, Jianfeng; Li, Yueqin; Jing, Chunxia; Zhou, Tianhong
The UL49 ORF of human cytomegalovirus (HCMV) is essential for viral replication; conserved among all herpes viruses; however, the function is unclear. Once the UL49 ORF was precisely deleted from the start to stop codon, the mutant did not yield infectious progeny. In this study, we find out many alternatively processed ESTs in UL49 locus in HCMV-infected cells, in which there are two novel transcription termination sites in UL49 locus. Most of these ESTs are rare transcripts that contain directed repeat sequences in the intron splicing regions. There is a typical GU-AG intron splicing site in UL49Y transcripts. The 1847 bp UL49Y cDNA spans an ORF from 335 to 1618 and encodes a putative protein of 427 amino acids with a predicted molecular mass of 47.1 kDa. All the new EST sequences and UL49Y cDNA sequence have been deposited in the GenBank database (GenBank Accession nos. GW314860-GW314900 and GU376796). This study provides us with very important clues for revealing the importance of the UL49 locus alternative splicing. PMID:25866769
Spatial mapping of splicing factor complexes involved in exon and intron definition
Ellis, Jonathan D.; Ll�res, David; Denegri, Marco; Lamond, Angus I.; C�ceres, Javier F.
We have analyzed the interaction between serine/arginine-rich (SR) proteins and splicing components that recognize either the 5? or 3? splice site. Previously, these interactions have been extensively characterized biochemically and are critical for both intron and exon definition. We use fluorescence resonance energy transfer (FRET) microscopy to identify interactions of individual SR proteins with the U1 small nuclear ribonucleoprotein (snRNP)�associated 70-kD protein (U1 70K) and with the small subunit of the U2 snRNP auxiliary factor (U2AF35) in live-cell nuclei. We find that these interactions occur in the presence of RNA polymerase II inhibitors, demonstrating that they are not exclusively cotranscriptional. Using FRET imaging by means of fluorescence lifetime imaging microscopy (FLIM), we map these interactions to specific sites in the nucleus. The FLIM data also reveal a previously unknown interaction between HCC1, a factor related to U2AF65, with both subunits of U2AF. Spatial mapping using FLIM-FRET reveals differences in splicing factors interactions within complexes located in separate subnuclear domains. PMID:18559666
Interactions of SR45, an SR-like protein, with spliceosomal proteins and an intronic sequence: insights into regulated splicing.
Day, Irene S; Golovkin, Maxim; Palusa, Saiprasad G; Link, Alicia; Ali, Gul S; Thomas, Julie; Richardson, Dale N; Reddy, Anireddy S N
SR45 is a serine/arginine-rich (SR)-like protein with two arginine/serine-rich (RS) domains. We have previously shown that SR45 regulates alternative splicing (AS) by differential selection of 5' and 3' splice sites. However, it is unknown how SR45 regulates AS. To gain mechanistic insights into the roles of SR45 in splicing, we screened a yeast two-hybrid library with SR45. This screening resulted in the isolation of two spliceosomal proteins, U1-70K and U2AF(35) b that are known to function in 5' and 3' splice site selection, respectively. This screen not only confirmed our prior observation that U1-70K and SR45 interact, but also helped to identify an additional interacting partner (U2AF(35) ). In vitro and in vivo analyses revealed an interaction of SR45 with both paralogs of U2AF(35) . Furthermore, we show that the RS1 and RS2 domains of SR45, and not the RNA recognition motif (RRM) domain, associate independently with both U2AF(35) proteins. Interaction studies among U2AF(35) paralogs and between U2AF(35) and U1-70K revealed that U2AF(35) can form homo- or heterodimers and that U2AF(35) proteins can associate with U1-70K. Using RNA probes from SR30 intron 10, whose splicing is altered in the sr45 mutant, we show that SR45 and U2AF(35) b bind to different parts of the intron, with a binding site for SR45 in the 5' region and two binding regions, each ending with a known 3' splice site, for U2AF(35) b. These results suggest that SR45 recruits U1snRNP and U2AF to 5' and 3' splice sites, respectively, by interacting with pre-mRNA, U1-70K and U2AF(35) and modulates AS. PMID:22563826
Carbon as a Shallow Donor in Transparent Conducting Oxides
NASA Astrophysics Data System (ADS)
Lyons, J. L.; Steiauf, D.; Janotti, A.; Van de Walle, C. G.
Carbon is a common unintentional impurity in oxide semiconductors. We use hybrid density functional theory to calculate the electronic and structural properties of carbon impurities in ZnO, In2O3 , and Ga2O3 �materials that are used as transparent conductors. In each of these semiconducting oxides, we find that carbon is most likely to occupy the cation site under most electronic and chemical potential conditions. In ZnO, CZn acts as a shallow double donor and exhibits large local breathing-mode relaxations. In In2O3 and Ga2O3 , C acts as a shallow donor and moves off the cation site to become threefold oxygen coordinated. In all three oxides, Ccation exhibits modest formation energies, indicating that these species will be likely to incorporate. Our results indicate that C impurities are suitable donor dopants in these oxides and will contribute to background n -type conductivity if unintentionally present.
The functional role of alternative splicing of Ca(2+)-activated K+ channels in auditory hair cells.
Jones, E M; Gray-Keller, M; Art, J J; Fettiplace, R
Turtle auditory hair cells are frequency tuned by the activity of large-conductance calcium-activated potassium (KCa) channels, the frequency range being dictated primarily by the channel kinetics. Seven alternatively spliced isoforms of the KCa channel alpha-subunit, resulting from exon insertion at two splice sites, were isolated from turtle hair cells. These, when expressed in Xenopus oocytes, produced KCa channels with a range of apparent calcium sensitivities and channel kinetics. However, most expressed channels were less calcium sensitive than the hair cells' native KCa channels. Coexpression of alpha-subunit with a bovine beta-subunit substantially increased the channel's calcium sensitivity while markedly slowing its kinetics, but kinetic differences between isoforms were preserved. These data suggest a molecular mechanism for hair cell frequency tuning involving differential expression of different KCa channel alpha-subunits in conjunction with an expression gradient of a regulatory beta-subunit. PMID:10414307
Select utilization of obese donors in living donor liver transplantation: implications for the donor pool.
Moss, J; Lapointe-Rudow, D; Renz, J F; Kinkhabwala, M; Dove, L M; Gaglio, P J; Emond, J C; Brown, R S
Living donor liver transplantation evolved in response to donor shortage. Current guidelines recommend potential living donors (LD) have a body mass index (BMI) <30. With the current obesity epidemic, locating nonobese LD is difficult. From September 1999 to August 2003, 68 LD with normal liver function test (LFTs) and without significant comorbidities underwent donor hepatectomy at our center. Post-operative complications were collected, including wound infection, pneumonia, hernia, fever, ileus, biliary leak, biliary stricture, thrombosis, bleeding, hepatic dysfunction, thrombocytopenia, deep venous thrombosis, pulmonary embolism, difficult to control pain, depression and anxiety. Complication rates for LD with BMI >30 (n = 16) and BMI <30 (n = 52) were compared. The incidence of wound infection increased with BMI, 4% for nonobese and 25% for obese LD (p = 0.024). There were no statistically significant differences for all other complications. No LD died. Recipient survival was 100% with obese LD and 80% with nonobese LD (p = 0.1). Select donors with a BMI >30 may undergo donor hepatectomy with acceptable morbidity and excellent recipient results. Updating current guidelines to include select LD with BMI >30 has the potential to safely increase the donor pool. PMID:16303013
RON alternative splicing regulation in primary ovarian cancer.
Mayer, Sebastian; Hirschfeld, Marc; Jaeger, Markus; Pies, Susanne; Iborra, Severine; Erbes, Thalia; Stickeler, Elmar
The proto-oncogene recepteur d'origine nantais (RON, MST1R) and its alternatively spliced variants are involved in various tumor biological processes, such as cell motility, adhesion, proliferation, apoptosis and epithelial-to-mesenchymal transition (EMT). RON overexpression and the occurrence of specific alternatively spliced RON isoforms have been detected in ovarian cancer. In the present study, we evaluated the role and regulation of cancer-related RON splicing isoforms in primary ovarian cancer. Expression of RON variants (RON?165, RON?160) was determined in 45 primary ovarian cancer and 4 physiological ovarian tissue specimens by RT-PCR and western blot analysis. The results were correlated to clinicopathological parameters. Additionally, expression of splicing factors with known involvement in RON alternative splicing regulation was examined. Increased RON levels were detected in all tumor samples (p=0.001) without differences between the primary tumors and metastases. Alternative RON variants were present in the majority of tumor samples (39 of 45; 86.67%). Potential RON?165 occurred more often (82.22%) than potential RON?160 or RON?155 (24.40%). Several significant correlations of RON and splicing factor expression [e.g. ASF/SFRS1 (p=0.035)] were detected. Correlations of RON expression to clinicopathological parameters were not observed. Significant splicing factor interactions (e.g. SRp55/SRp75: p<0.001) were observed in tumor samples with alternative RON splicing. Our data demonstrated upregulated RON isoform expression and significant changes in splicing factor expression in primary ovarian cancer. These findings account for an essential regulatory interplay of splicing factor-driven alterations in the RON alternative splicing pattern with subsequent tumor biological consequences in ovarian cancer. PMID:25997828
Donor Matching for Allogenic Transplant
... Registry . They have access to millions of international records, and have successfully matched thousands of donors and ... single match can require going through millions of records. Now that transplant centers are more often using ...
Donor selection in heart transplantation
Emani, Sitaramesh; Sai-Sudhakar, Chittoor B.; Higgins, Robert S. D.; Whitson, Bryan A.
There is increased scrutiny on the quality in health care with particular emphasis on institutional heart transplant survival outcomes. An important aspect of successful transplantation is appropriate donor selection. We review the current guidelines as well as areas of controversy in the selection of appropriate hearts as donor organs to ensure optimal outcomes. This decision is paramount to the success of a transplant program as well as recipient survival and graft function post-transplant. PMID:25132976
Complications of Laparoscopic Donor Nephrectomy
Alexei Wedmid; Michael A. Palese
\\u000a �Laparoscopic donor nephrectomy is a unique surgical procedure due to the fact that the surgeon is operating on a healthy\\u000a individual in order to benefit another patient he or she is unlikely managing, with a potential for complications ensuing\\u000a in both the donor and the recipient patients. Overall surgical technique, anatomic considerations, and perioperative management\\u000a remain important for minimizing the
Mass transfer from giant donors
NASA Astrophysics Data System (ADS)
Pavlovskii, K.; Ivanova, N.
The stability of mass transfer in binaries with convective giant donors remains an open question in modern astrophysics. There is a significant discrepancy between what the existing methods predict for a response to mass-loss of the giant itself, as well as for the mass-transfer rate during the Roche lobe overflow. Here we show that the recombination energy in the superadiabatic layer plays an important and hitherto unaccounted-for role in the donor's response to mass-loss, in particular on its luminosity and effective temperature. Our improved optically thick nozzle method to calculate the mass-transfer rate via L1 allows us to evolve binary systems for a substantial Roche lobe overflow. We propose a new, strengthened criterion for the mass-transfer instability, basing it on whether the donor experiences overflow through its outer Lagrangian point. We find that with the new criterion, if the donor has a well-developed outer convective envelope, the critical initial mass ratio for which a binary would evolve stably through the conservative mass transfer varies from 1.5 to 2.2, which is about twice as large as previously believed. In underdeveloped giants with shallow convective envelopes, this critical ratio may be even larger. When the convective envelope is still growing, and in particular for most cases of massive donors, the critical mass ratio gradually decreases to this value, from that of radiative donors.
Persistency and permanency of two stages DNA splicing languages with respect to one initial string and two rules via Yusof-Goode (Y-G) approach
NASA Astrophysics Data System (ADS)
Mudaber, Mohammad Hassan; Yusof, Yuhani; Mohamad, Mohd Sham; Lim, Wen Li
The notion of Yusof-Goode (Y-G) splicing system was first schemed by Yusof to study the relationship between formal language theory and molecular biology. The splicing languages that are produced by splicing system have some important characteristics called persistent and permanent. In biological perspective, the recombinant DNA molecules can be manipulated by recombination action if they have persistent property. Thus, the persistency as well as permanency of splicing languages (recombinant DNA molecules) is considered to be an interesting topic in the field of DNA recombination, particularly when the recombination process is accomplished at second stage. Conducting a wet-lab experiment to show the mentioned properties of splicing languages are time consuming and expensive. Therefore, to overcome this problem, mathematical approach is chosen to investigate the persistency and permanency of splicing languages which will be then given as theorem and corollary. Thus, an initial string (with two recognition sites) and two rules are considered for introducing the above characteristics using Y-G approach.
Structure of phosphorylated SF1 bound to U2AF65 in an essential splicing factor complex
Wang, Wenhua; Maucuer, Alexandre; Gupta, Ankit; Manceau, Val�rie; Thickman, Karen R.; Bauer, William J.; Kennedy, Scott D.; Wedekind, Joseph E.; Green, Michael R.; Kielkopf, Clara L.
SUMMARY The essential splicing factors U2AF65 and SF1 cooperatively bind consensus sequences at the 3? end of introns. Phosphorylation of SF1 on a highly conserved �SPSP� motif enhances its interaction with U2AF65 and the pre-mRNA. Here we reveal that phosphorylation-induces essential conformational changes in SF1 and in the SF1/U2AF65/3? splice site complex. Crystal structures of the phosphorylated (P)SF1 domain bound to the C-terminal domain of U2AF65 at 2.29 � resolution, and of the unphosphorylated SF1 domain at 2.48 � resolution, demonstrate that phosphorylation induces a disorder-to-order transition within a novel SF1/U2AF65 interface. We find by small-angle X-ray scattering that the local folding of the SPSP motif transduces into global conformational changes in the nearly full length (P)SF1/U2AF65/3? splice site assembly. We further determine that SPSP phosphorylation and the novel SF1/U2AF65 interface are essential in vivo. These results offer a structural prototype for phosphorylation-dependent control of pre-mRNA splicing factors. PMID:23273425
RASA: Robust Alternative Splicing Analysis for Human Transcriptome Arrays.
Seok, Junhee; Xu, Weihong; Davis, Ronald W; Xiao, Wenzhong
Human transcriptome arrays (HTA) have recently been developed for high-throughput alternative splicing analysis by measuring signals not only from exons but also from exon-exon junctions. Effective use of these rich signals requires the development of computational methods for better gene and alternative splicing analyses. In this work, we introduce a computational method, Robust Alternative Splicing Analysis (RASA), for the analysis of the new transcriptome arrays by effective integration of the exon and junction signals. To increase robustness, RASA calculates the expression of each gene by selecting exons classified as not alternatively spliced. It then identifies alternatively spliced exons that are supported by both exon and junction signals to reduce the false positives. Finally, it detects additional alternative splicing candidates that are supported by only exon signals because the signals from the corresponding junctions are not well detected. RASA was demonstrated with Affymetrix HTAs and its performance was evaluated with mRNA-Seq and RT-PCR. The validation rate is 52.4%, which is a 60% increase when compared with previous methods that do not use selected exons for gene expression calculation and junction signals for splicing detection. These results suggest that RASA significantly improves alternative splicing analyses on HTA platforms. PMID:26145443
Scattering of acoustic duct modes by axial liner splices
NASA Astrophysics Data System (ADS)
Tam, Christopher K. W.; Ju, Hongbin; Chien, Eugene W.
Recent engine test data and results of computational analysis show that the engine inlet acoustic liner splices have a significant impact on aircraft flight noise certification and cabin noise levels. The phenomenon of scattering of acoustic duct modes by axial liner splices is investigated. Previous studies, invariably, follow the frequency-domain approach. The present study, however, uses the time-domain approach. It is demonstrated that time-domain computation yields results that are in close agreement with frequency-domain results. The scattering phenomenon under consideration is very complex. This study concentrates on the effects of four parameters. They are the width of the splices, the frequency of the incident duct mode, the number of splices and the length of splices. Based on the computed results, the conditions under which scattered wave modes would significantly increase the intensity of transmitted waves are identified. It is also found that surface scattering by liner splices has the tendency to distribute energy equally to all the cut-on scattered azimuthal modes. On the other hand, for each scattered azimuthal mode, the high-order cut-on radial mode, generally, has the highest intensity. Moreover, scattering by liner splices is a local phenomenon. It is confined primarily to an area of the duct adjacent to the junction between the hard wall near the fan face and the spliced liner.
HIFs Enhance the Transcriptional Activation and Splicing of Adrenomedullin
Sena, Johnny A.; Wang, Liyi; Pawlus, Matthew R.; Hu, Cheng-Jun
Adrenomedullin (ADM) is important for tumor angiogenesis, tumor cell growth and survival. Under normoxic conditions, the ADM gene was found to produce two alternative transcripts, a fully-spliced transcript that produces AM and PAMP peptides and a intron-3-retaining transcript that produces a less functionally significant PAMP peptide only. ADM is a well-established hypoxia inducible gene; however, it is not clear which ADM isoform is induced by hypoxia. In this study, it was determined that various cancer and normal cells express two predominant types of ADM transcripts, a AM/PAMP peptide producing FL transcript in which all introns are removed, and a non-protein producing I1-3 transcript in which all introns are retained. Interestingly, hypoxia preferentially induced the FL isoform. Moreover, HIFs, but not hypoxia per se are necessary and sufficient to increase splicing of ADM pre-mRNA. ADM splicing reporters confirmed that transcriptional activation by HIF or other transcription factors is sufficient to enhance splicing. However, HIFs are more potent in enhancing ADM pre-mRNA splicing than other transcriptional activators. Thus, ADM intron retention is not a consequence of abnormal splicing, but is an important mechanism to regulate ADM expression. These results demonstrate a novel function of HIFs in regulating ADM expression by enhancing its pre-mRNA splicing. Importantly, using endogenous and cloned ADM gene, further evidence is provided for the coupling of transcription and RNA splicing. PMID:24523299
The importance of being divisible by three in alternative splicing
Ast, Gil
The importance of being divisible by three in alternative splicing Alon Magen and Gil Ast* Department of Human Genetics and Molecular Medicine, Sackler Faculty of Medicine, Tel Aviv University, Ramat Alternative splicing events that are conserved in orthologous genes in different species are commonly viewed
Global analysis of alternative splicing differences between humans and chimpanzees
Calarco, John A.; Xing, Yi; C�ceres, Mario; Calarco, Joseph P.; Xiao, Xinshu; Pan, Qun; Lee, Christopher; Preuss, Todd M.; Blencowe, Benjamin J.
Alternative splicing is a powerful mechanism affording extensive proteomic and regulatory diversity from a limited repertoire of genes. However, the extent to which alternative splicing has contributed to the evolution of primate species-specific characteristics has not been assessed previously. Using comparative genomics and quantitative microarray profiling, we performed the first global analysis of alternative splicing differences between humans and chimpanzees. Surprisingly, 6%�8% of profiled orthologous exons display pronounced splicing level differences in the corresponding tissues from the two species. Little overlap is observed between the genes associated with alternative splicing differences and the genes that display steady-state transcript level differences, indicating that these layers of regulation have evolved rapidly to affect distinct subsets of genes in humans and chimpanzees. The alternative splicing differences we detected are predicted to affect diverse functions including gene expression, signal transduction, cell death, immune defense, and susceptibility to diseases. Differences in expression at the protein level of the major splice variant of Glutathione S-transferase omega-2 (GSTO2), which functions in the protection against oxidative stress and is associated with human aging-related diseases, suggests that this enzyme is less active in human cells compared with chimpanzee cells. The results of this study thus support an important role for alternative splicing in establishing differences between humans and chimpanzees. PMID:17978102
Discovering Transcription and Splicing Networks in Myelodysplastic Syndromes
Wang, Hongyan; Wen, Jianguo; Chang, Chung-che; Zhou, Xiaobo
More and more transcription factors and their motifs have been reported and linked to specific gene expression levels. However, focusing only on transcription is not sufficient for mechanism research. Most genes, especially in eukaryotes, are alternatively spliced to different isoforms. Some of these isoforms increase the biodiversity of proteins. From this viewpoint, transcription and splicing are two of important mechanisms to modulate expression levels of isoforms. To integrate these two kinds of regulation, we built a linear regression model to select a subset of transcription factors and splicing factors for each co-expressed isoforms using least-angle regression approach. Then, we applied this method to investigate the mechanism of myelodysplastic syndromes (MDS), a precursor lesion of acute myeloid leukemia. Results suggested that expression levels of most isoforms were regulated by a set of selected regulatory factors. Some of the detected factors, such as EGR1 and STAT family, are highly correlated with progression of MDS. We discovered that the splicing factor SRSF11 experienced alternative splicing switch, and in turn induced different amino acid sequences between MDS and controls. This splicing switch causes two different splicing mechanisms. Polymerase Chain Reaction experiments also confirmed that one of its isoforms was over-expressed in MDS. We analyzed the regulatory networks constructed from the co-expressed isoforms and their regulatory factors in MDS. Many of these networks were enriched in the herpes simplex infection pathway which involves many splicing factors, and pathways in cancers and acute or chronic myeloid leukemia. PMID:24244432
Numerical analyses of splice losses of photonic crystal fibers
Zhongnan Xu; Kailiang Duan; Zejin Liu; Yishan Wang; Wei Zhao
Splice losses between a photonic crystal fiber (PCF) and a single mode fiber (SMF) or a PCF are numerically investigated by using finite element method (FEM) with the circular perfectly matched layer (PML). Results show that the splice loss between a SMF and a PCF with air holes completely collapsed can reach many times of that between a SMF and
Molecular Cell Tissue-Specific Splicing of Disordered
Babu, M. Madan
Molecular Cell Article Tissue-Specific Splicing of Disordered Segments that Embed Binding Motifs diversity of proteins. Among alternatively spliced exons, tissue-specific exons play a critical role in maintaining tissue identity. This raises the question of how tissue-specific protein-coding exons influence
Splicing Systems: Regularity and Below , Dennis Pixton1
Goode, Elizabeth
realizations of abstract character strings. The nucleic acids, proteins, and many additional polymers admit, the formal definition of splicing used here, which is essentially Gh. Paun's, has become standard. Let for some q, p in A }. For each set, R, of splicing rules we extend the definition of r(L) by defining R
A Trajectory Splicing Approach to Concretizing Counterexamples for Hybrid Systems
Sankaranarayanan, Sriram
for finding falsi- fying trajectories of hybrid systems using an approach that we call trajectory splicing. In practice, it is important to provide concrete error trajectories that can be examined by a control engineerA Trajectory Splicing Approach to Concretizing Counterexamples for Hybrid Systems Aditya Zutshi
Alternative splicing and biological heterogeneity in prostate cancer
David J. Elliott; Craig N. Robson; Hing Y. Leung; Prabhakar Rajan
The biological diversity of prostate cancer confounds standardization of therapy. Advances in molecular profiling suggest that differences in the genetic composition of tumors significantly contribute to the complexity of the disease. Alternative pre-mRNA splicing is a key genetic process underlying biological diversity. During alternative splicing, coding and noncoding regions of a single gene are rearranged to generate several messenger RNA
RASA: Robust Alternative Splicing Analysis for Human Transcriptome Arrays
Seok, Junhee; Xu, Weihong; Davis, Ronald W.; Xiao, Wenzhong
Human transcriptome arrays (HTA) have recently been developed for high-throughput alternative splicing analysis by measuring signals not only from exons but also from exon-exon junctions. Effective use of these rich signals requires the development of computational methods for better gene and alternative splicing analyses. In this work, we introduce a computational method, Robust Alternative Splicing Analysis (RASA), for the analysis of the new transcriptome arrays by effective integration of the exon and junction signals. To increase robustness, RASA calculates the expression of each gene by selecting exons classified as not alternatively spliced. It then identifies alternatively spliced exons that are supported by both exon and junction signals to reduce the false positives. Finally, it detects additional alternative splicing candidates that are supported by only exon signals because the signals from the corresponding junctions are not well detected. RASA was demonstrated with Affymetrix HTAs and its performance was evaluated with mRNA-Seq and RT-PCR. The validation rate is 52.4%, which is a 60% increase when compared with previous methods that do not use selected exons for gene expression calculation and junction signals for splicing detection. These results suggest that RASA significantly improves alternative splicing analyses on HTA platforms. PMID:26145443
Alternative splicing and evolution: diversification, exon definition and function
Galit Lev-Maor; Gil Ast; Hadas Keren
Over the past decade, it has been shown that alternative splicing (AS) is a major mechanism for the enhancement of transcriptome and proteome diversity, particularly in mammals. Splicing can be found in species from bacteria to humans, but its prevalence and characteristics vary considerably. Evolutionary studies are helping to address questions that are fundamental to understanding this important process: how
Insights into alternative splicing of sarcomeric genes in the heart.
Weeland, Cornelis J; van den Hoogenhof, Maarten M; Beqqali, Abdelaziz; Creemers, Esther E
Driven by rapidly evolving technologies in next-generation sequencing, alternative splicing has emerged as a crucial layer in gene expression, greatly expanding protein diversity and governing complex biological processes in the cardiomyocyte. At the core of cardiac contraction, the physical properties of the sarcomere are carefully orchestrated through alternative splicing to fit the varying demands on the heart. By the recent discovery of RBM20 and RBM24, two major heart and skeletal muscle-restricted splicing factors, it became evident that alternative splicing events in the heart occur in regulated networks rather than in isolated events. Analysis of knockout mice of these splice factors has shed light on the importance of these fundamental processes in the heart. In this review, we discuss recent advances in our understanding of the role and regulation of alternative splicing in the developing and diseased heart, specifically within the sarcomere. Through various examples (titin, myomesin, troponin T, tropomyosin and LDB3) we illustrate how alternative splicing regulates the functional properties of the sarcomere. Finally, we evaluate opportunities and obstacles to modulate alternative splicing in therapeutic approaches for cardiac disease. PMID:25683494
Intronic Alus Influence Alternative Splicing Galit Lev-Maor1.
Ast, Gil
in the primate-specific retrotransposed element called Alu. A large fraction of Alus are found in intronic Alus on splicing of the flanking exons is largely unknown. Here, we show that more Alus flank-pairing, as evident by RNA editing, and affect the splicing patterns of a downstream exon, shifting it from
Automated fusion-splicing of polarization maintaining fibers
Wenzin Zheng
An advanced splicing technique for polarization maintaining (PM) fibers has been derived based on the polarization observation by lens-effect-tracing (POL) method. With this technique, azimuthal alignment on common types of PM fibers can be automatically performed in a passive way by an automated fusion splicer. Because the method permits an accurate estimation of the splice's extinction ratio before and after
The changing profile of the cardiac donor
Malcolm V Brock; Jorge D Salazar; Duke E Cameron; William A Baumgartner; John V Conte
Background: Expansion of traditional donor criteria has become standard in most centers. To determine how this has affected donor profiles, at our institution, we reviewed all adult (age ? 16) cardiac donors of the past 15 years.Methods: We separated 261 cardiac donors into 2 groups based on time periods: Group I, 1983 to 1991 (n = 131), and Group II,
Blood Donation by Elderly Repeat Blood Donors
Zeiler, Thomas; Lander-Kox, Jutta; Alt, Timo
Summary Background Upper age limits for blood donors are intended to protect elderly blood donors from donor reactions. However, due to a lack of data about adverse reactions in elderly blood donors, upper age limits are arbitrary and vary considerably between different countries. Methods Here we present data from 171,231 voluntary repeat whole blood donors beyond the age of 68 years. Results Blood donations from repeat blood donors beyond the age of 68 years increased from 2,114 in 2005 to 38,432 in 2012 (from 0,2% to 4.2% of all whole blood donations). Adverse donor reactions in repeat donors decreased with age and were lower than in the whole group (0.26%), even in donors older than 71 years (0.16%). However, from the age of 68 years, the time to complete recovery after donor reactions increased. Donor deferrals were highest in young blood donors (21.4%), but increased again in elderly blood donors beyond 71 years (12.6%). Conclusion Blood donation by regular repeat blood donors older than 71 years may be safely continued. However, due to a lack of data for donors older than 75 years, blood donation in these donors should be handled with great caution. PMID:25254019
Natural Variation in the Drosophila melanogaster Clock Gene Period Modulates Splicing of Its 3?-Terminal Intron and Mid-Day Siesta
Low, Kwang Huei; Chen, Wen-Feng; Yildirim, Evrim; Edery, Isaac
Drosophila melanogaster exhibits circadian (?24 hr) regulated morning and evening bouts of activity that are separated by a mid-day siesta. Increases in daily ambient temperature are accompanied by a progressively longer mid-day siesta and delayed evening activity. Presumably, this behavioral plasticity reflects an adaptive response that endows D. melanogaster with the ability to temporally optimize daily activity levels over a wide range of physiologically relevant temperatures. For example, the shift in activity towards the cooler nighttime hours on hot days might minimize the risks associated with exposure to mid-day heat, whereas on cold days activity is favored during the warmer daytime hours. These temperature-induced shifts in the distribution of daily activity are partly based on the thermal sensitive splicing of an intron found in the 3? untranslated region (UTR) of the circadian clock gene termed period (per). As temperature decreases, splicing of this 3?-terminal intron (termed dmpi8) is gradually increased, which is causally linked to a shorter mid-day siesta. Herein we identify several natural polymorphisms in the per 3? UTR from wild-caught populations of flies originating along the east coast of the United States. Two non-intronic closely spaced single nucleotide polymorphisms (SNPs) modulate dmpi8 splicing efficiency, with the least efficiently spliced version associated with a longer mid-day siesta, especially at lower temperatures. Although these SNPs modulate the splicing efficiency of dmpi8 they have little to no effect on its thermal responsiveness, consistent with the notion that the suboptimal 5? and 3? splice sites of the dmpi8 intron are the primary cis-acting elements mediating temperature regulation. Our results demonstrate that natural variations in the per gene can modulate the splicing efficiency of the dmpi8 intron and the daily distribution of activity, providing natural examples for the involvement of dmpi8 splicing in the thermal adaptation of behavioral programs in D. melanogaster. PMID:23152918
Functional and splicing defect analysis of 23 ACVRL1 mutations in a cohort of patients affected by Hereditary Hemorrhagic Telangiectasia
Alaa el Din, Ferdos; Patri, Sylvie; Thoreau, Vincent; Rodriguez-Ballesteros, Montserrat; Hamade, Eva; Bailly, Sabine; Gilbert-Dussardier, Brigitte; Abou Merhi, Raghida; Kitzis, Alain
Hereditary Hemorrhagic Telangiectasia syndrome (HHT) or Rendu-Osler-Weber (ROW) syndrome is an autosomal dominant vascular disorder. Two most common forms of HHT, HHT1 and HHT2, have been linked to mutations in the endoglin (ENG) and activin receptor-like kinase 1 (ACVRL1or ALK1) genes respectively. This work was designed to examine the pathogenicity of 23 nucleotide variations in ACVRL1 gene detected in more than 400 patients. Among them, 14 missense mutations and one intronic variant were novels, and 8 missense mutations were previously identified with questionable implication in HHT2. The functionality of missense mutations was analyzed in response to BMP9 (specific ligand of ALK1), the maturation of the protein products and their localization were analyzed by western blot and fluorescence microscopy. The splicing impairment of the intronic and of two missense mutations was examined by minigene assay. Functional analysis showed that 18 out of 22 missense mutations were defective. Splicing analysis revealed that one missense mutation (c.733A>G, p.Ile245Val) affects the splicing of the harboring exon 6. Similarly, the intronic mutation outside the consensus splicing sites (c.1048+5G>A in intron 7) was seen pathogenic by splicing study. Both mutations induce a frame shift creating a premature stop codon likely resulting in mRNA degradation by NMD surveillance mechanism. Our results confirm the haploinsufficiency model proposed for HHT2. The affected allele of ACVRL1 induces mRNA degradation or the synthesis of a protein lacking the receptor activity. Furthermore, our data demonstrate that functional and splicing analyses together, represent two robust diagnostic tools to be used by geneticists confronted with novel or conflicted ACVRL1 mutations. PMID:26176610
Who is the best alternative allotransplant donor?
Gale, R P; Eapen, M
Assuming that most physicians will chose an HLA-identical sibling as the best allotransplant donor, the question arises who is the best alternative donor when an HLA-identical sibling is unavailable? The most commonly used alternative donors are HLA-identical or -mismatched unrelated donors, HLA-matched or -mismatched umbilical cord blood donor or a related, HLA-haplotype-matched related donors. Each alternative donor option has advantages and disadvantages. We discuss selected aspects of these issues based on data from randomized clinical trials and observational databases. However, because there are limited data to address specific clinical settings, quantification of expert opinion is sometimes needed. PMID:26039206
Detecting Image Splicing Using Merged Features in Chroma Space
Liu, Guangjie; Dai, Yuewei
Image splicing is an image editing method to copy a part of an image and paste it onto another image, and it is commonly followed by postprocessing such as local/global blurring, compression, and resizing. To detect this kind of forgery, the image rich models, a feature set successfully used in the steganalysis is evaluated on the splicing image dataset at first, and the dominant submodel is selected as the first kind of feature. The selected feature and the DCT Markov features are used together to detect splicing forgery in the chroma channel, which is convinced effective in splicing detection. The experimental results indicate that the proposed method can detect splicing forgeries with lower error rate compared to the previous literature. PMID:24574877
The Electrical Resistance of Rutherford-Type Superconducting Cable Splices
Heck, C; Fleiter, J; Bottura, L
The electrical resistance of Large Hadron Collider main busbar cable lap splices produced by soft soldering has been measured with two independent methods as a function of intercable contact area and for splices made of cables with various defects. For defect-free lap splices, the resistance increases from 0.3 to 10 n? (at 4.3 K in self-field) when reducing the cable overlap length from 120 to 3 mm, as expected assuming that the resistance is inversely proportional to the intercable contact area. The resistance of bridge splices that connect side-by-side cables can be predicted from the lap splice resistances and the overlap areas involved.
[Alternative splicing regulation: implications in cancer diagnosis and treatment].
Mart�nez-Montiel, Nancy; Rosas-Murrieta, Nora; Mart�nez-Contreras, Rebeca
The accurate expression of the genetic information is regulated by processes like mRNA splicing, proposed after the discoveries of Phil Sharp and Richard Roberts, who demonstrated the existence of intronic sequences, present in almost every structural eukaryotic gene, which should be precisely removed. This intron removal is called "splicing", which generates different proteins from a single mRNA, with different or even antagonistic functions. We currently know that alternative splicing is the most important source of protein diversity, given that 70% of the human genes undergo splicing and that mutations causing defects in this process could originate up to 50% of genetic diseases, including cancer. When these defects occur in genes involved in cell adhesion, proliferation and cell cycle regulation, there is an impact on cancer progression, rising the opportunity to diagnose and treat some types of cancer according to a particular splicing profile. PMID:24725854
Branched Intermediate Formation Stimulates Peptide Bond Cleavage in Protein Splicing
Frutos, Silvia; Goger, Michael; Giovani, Baldissera; Cowburn, David; Muir, Tom W.
Protein splicing is a posttranslational modification in which an intein domain excises itself out of a host protein. Here, we investigate how the steps in the splicing process are coordinated so as to maximize the production of the final splice products and minimize the generation of undesired cleavage products. Our approach has been to prepare a branched intermediate (and analogs thereof) of the Mxe GyrA intein using protein semi-synthesis. Kinetic analysis of these molecules indicates that the high fidelity of this protein splicing reaction results from the penultimate step in the process (intein-succinimide formation) being rate-limiting. NMR experiments indicate that formation of the branched intermediate affects the local structure around the amide bond cleaved during succinimide formation. We propose that this structural change reflects a re-organization of the catalytic apparatus to accelerate succinimide formation at the C-terminal splice junction. PMID:20495572
Should pharmacologists care about alternative splicing? IUPHAR Review 4
Bonner, T I
Alternative splicing of mRNAs occurs in the majority of human genes, and most differential splicing results in different protein isoforms with possibly different functional properties. However, there are many reported splicing variations that may be quite rare, and not all combinatorially possible variants of a given gene are expressed at significant levels. Genes of interest to pharmacologists are frequently expressed at such low levels that they are not adequately represented in genome-wide studies of transcription. In single-gene studies, data are commonly available on the relative abundance and functional significance of individual alternatively spliced exons, but there are rarely data that quantitate the relative abundance of full-length transcripts and define which combinations of exons are significant. A number of criteria for judging the significance of splice variants and suggestions for their nomenclature are discussed. PMID:24670145
Electron Spin Resonance Experiments on Donors in Silicon. I. Electronic Structure of Donors by the Electron Nuclear Double Resonance Technique
G. Feher
The ground-state wave function of the antimony, phosphorus, and arsenic impurities in silicon has been investigated by means of the electron nuclear double resonance (ENDOR) method. By this method the hyperfine interactions of the donor electron with the Si29 nuclei situated at different lattice sites were obtained. The isotropic part of the hyperfine interaction agreed with the theory of Kohn
Electron Donor Potential of Eastern North Dakota Shale Formations
R. J. Salinas Klapperich; S. F. Korom
We have a network of 16 in situ mesocosms (ISMs) used to study aquifer denitrification at 9 sites in North Dakota and Minnesota. The site in the Elk Valley aquifer in northeastern North Dakota has the highest denitrification rates and the greatest concentration of electron donors (organic carbon ~0.4%, pyrite as S ~0.4%, and ferrous iron ~0.3%) in the sediments.
A generalized dSpliceType framework to detect differential splicing and differential expression events using RNA-Seq.
Zhu, Dongxiao; Deng, Nan; Bai, Changxin
Transcriptomes are routinely compared in term of a list of differentially expressed genes followed by functional enrichment analysis. Due to the technology limitations of microarray, the molecular mechanisms of differential expression is poorly understood. Using RNA-seq data, we propose a generalized dSpliceType framework to systematically investigate the synergistic and antagonistic effects of differential splicing and differential expression. We applied the method to two public RNA-seq data sets and compared the transcriptomes between treatment and control conditions. The generalized dSpliceType detects and prioritizes a list of genes that are differentially expressed and/or spliced. In particular, the multivariate dSpliceType is among the fist to utilize sequential dependency of normalized base-wise read coverage signals and capture biological variability among replicates using a multivariate statistical model. We compared dSpliceType with two other methods in terms of five most common types of differential splicing events between two conditions using RNA-Seq. dSpliceType is free, available from http://dsplicetype.sourceforge.net/. PMID:25680210
Donor height in combination with islet donor score improves pancreas donor selection for pancreatic islet isolation and transplantation.
Wang, L J; Cochet, O; Wang, X J; Krzystyniak, A; Misawa, R; Golab, K; Tibudan, M; Grose, R; Savari, O; Millis, J M; Witkowski, P
To maximize the islet isolation yield for successful islet transplantation, the key task has been to identify an ideal pancreas donor. Since implementation of the islet donor score in donor selection, we have consistently obtained higher islet yields and transplantation rates. In this study, we tested whether assessing donor height as an independent variable in combination with the donor score could improve the pancreas donor selection. Donor and islet isolation information (n�= 22) were collected and studied between 2011 and 2012. Pearson correlation analysis was used in statistical analysis. Donor height as an independent variable was significantly correlated to the weight of the pancreas, pre-Islet Equivalents (pre-IEQ), post-IEQ, and IDS (P�< .05). When donor with height of 179�cm � 3 was selected in combination with IDS > 80, the clinical islet transplantation rate reached 80%. PMID:25131085
2002-09 Sami Khuri Biology Terms and Model Organisms
Khuri, Sami
alternative splicing open reading frame promoter TATA-box operon e.coli baker's yeast arabidopsis drosophila c nucleic acid deoxyribonucleic acid: DNA ribonucleic acid: RNA central dogma of biology human genome expression splicing human genome project splice donor site splice acceptor site consensus sequence
Mis-splicing of the ABCC2 gene linked with Bt toxin resistance in Helicoverpa armigera
Xiao, Yutao; Zhang, Tao; Liu, Chenxi; Heckel, David G.; Li, Xianchun; Tabashnik, Bruce E.; Wu, Kongming
Toxins from the bacterium Bacillus thuringiensis (Bt) are used widely for insect control in sprays and transgenic plants, but their efficacy is reduced when pests evolve resistance. Previous work showed that mutations in a gene encoding the transporter protein ABCC2 are linked with resistance to Bt toxins Cry1Ab, Cry1Ac or both in four species of Lepidoptera. Here we compared the ABCC2 gene of Helicoverpa armigera (HaABCC2) between susceptible strains and a laboratory-selected strain with >1,000-fold resistance to Cry1Ac relative its susceptible parent strain. We discovered a 73-base pair (bp) insertion in the cDNA of the resistant strain that generates a premature stop codon expected to yield a truncated ABCC2 protein. Sequencing of genomic DNA revealed that this insertion is an intron that is not spliced out because of a 6-bp deletion at its splicing site. Analysis of progeny from crosses revealed tight genetic linkage between HaABCC2 and resistance to Cry1Ac. These results provide the first evidence that mis-splicing of a gene encoding an ABCC2 protein confers resistance to a Bt toxin. PMID:25154974
Fusion Splicing Photonic Crystal Fibers and Conventional Single-Mode Fibers: Microhole Collapse Effect
Limin Xiao; M. S. Demokan; Wei Jin; Yiping Wang; Chun-Liu Zhao
We investigate the microhole collapse property of different photonic crystal fibers (PCFs) and its effect on the splice loss using an electric arc fusion splicer. The physical mechanism of the splice loss for different kinds of PCFs is studied, and a guideline for splicing these PCFs and conventional single-mode fibers (SMFs) is proposed. We demonstrate a low-loss fusion splicing of
Aberrantly spliced HTT, a new player in Huntington�s disease pathogenesis
Gipson, Theresa A; Neueder, Andreas; Wexler, Nancy S; Bates, Gillian P; Housman, David
Huntington�s disease (HD) is an adult-onset neurodegenerative disorder caused by a mutated CAG repeat in the huntingtin gene that is translated into an expanded polyglutamine tract. The clinical manifestation of HD is a progressive physical, cognitive, and psychiatric deterioration that is eventually fatal. The mutant huntingtin protein is processed into several smaller fragments, which have been implicated as critical factors in HD pathogenesis. The search for proteases responsible for their production has led to the identification of several cleavage sites on the huntingtin protein. However, the origin of the small N-terminal fragments that are found in HD postmortem brains has remained elusive. Recent mapping of huntingtin fragments in a mouse model demonstrated that the smallest N-terminal fragment is an exon 1 protein. This discovery spurred our hypothesis that mis-splicing as opposed to proteolysis could be generating the smallest huntingtin fragment. We demonstrated that mis-splicing of mutant huntingtin intron 1 does indeed occur and results in a short polyadenylated mRNA, which is translated into an exon 1 protein. The exon 1 protein fragment is highly pathogenic. Transgenic mouse models containing just human huntingtin exon 1 develop a rapid onset of HD-like symptoms. Our finding that a small, mis-spliced HTT transcript and corresponding exon 1 protein are produced in the context of an expanded CAG repeat has unraveled a new molecular mechanism in HD pathogenesis. Here we present detailed models of how mis-splicing could be facilitated, what challenges remain in this model, and implications for therapeutic studies. PMID:24256709
64 FR 34260 - Blood Donor Suitability Workshop: Donor History of Hepatitis
Federal Register 2010, 2011, 2012, 2013, 2014
...Suitability Workshop: Donor History of Hepatitis AGENCY: Food and Drug Administration...Suitability Workshop: Donor History of Hepatitis.'' The purpose of the workshop is...blood donors with a history of viral hepatitis should be deferred from donating...
The Unusual 23S rRNA Gene of Coxiella burnetii: Two Self-Splicing Group I Introns Flank a 34-Base-Pair Exon, and One Element Lacks the Canonical �G
Rahul Raghavan; Scott R. Miller; Linda D. Hicks; Michael F. Minnick
We describe the presence and characteristics of two self-splicing group I introns in the sole 23S rRNA gene of Coxiella burnetii. The two group I introns, Cbu.L1917 and Cbu.L1951, are inserted at sites 1917 and 1951 (Escherichia coli numbering), respectively, in the 23S rRNA gene of C. burnetii. Both introns were found to be self-splicing in vivo and in vitro
Cloning of the Full-Length Coding Sequence of Rat Liver-Specific Organic Anion Transporter1 (rlst-1) and a Splice Variant and Partial Characterization of the Rat lst-1 Gene
Supratim Choudhuri; Kenichiro Ogura; Curtis D. Klaassen
The full-length coding sequence of rat liver-specific organic anion transporter-1 (lst-1) and its splice variant have been cloned. The full-length rat lst-1 (designated rlst-1a) encodes a protein containing 687 amino acids and has 12-putative transmembrane domains, multiple potential N-glycosylation and phosphorylation sites. Therefore, rat lst-1a has 35 additional amino acid residues compared to the previously reported rat lst-1. A splice
Predicting Splice Variant from DNA Chip Expression Data
Hu, Gang Ken; Madore, Steven J; Moldover, Brian; Jatkoe, Tim; Balaban, David; Thomas, Jeffrey; Wang, Yixin
Alternative splicing of premessenger RNA is an important layer of regulation in eukaryotic gene expression. Splice variation of a large number of genes has been implicated in various cell growth and differentiation processes. To measure tissue-specific splicing of genes on a large scale, we collected gene expression data from 11 rat tissues using a high-density oligonucleotide array representing 1600 rat genes. Expression of each gene on the chip is measured by 20 pairs of independent oligonucleotide probes. Two algorithms have been developed to normalize and compare the chip hybridization signals among different tissues at individual oligonucleotide probe level. Oligonucleotide probes (the perfect match [PM] probe of each probe pair), detecting potential tissue-specific splice variants, were identified by the algorithms. The identified candidate splice variants have been compared to the alternatively spliced transcripts predicted by an EST clustering program. In addition, 50% of the top candidates predicted by the algorithms were confirmed by RT-PCR experiment. The study indicates that oligonucleotide probe-based DNA chip assays provide a powerful approach to detect splice variants at genome scale. PMID:11435406
The Resistance and Strength of Soft Solder Splices between Conductors in MICE Coils
Wu, Hong; Pan, Heng; Green, Michael A; Dietderich, Dan; Gartner, T. E.; Higley, Hugh C; Mentink, M.; Xu, FengYu; Trillaud, F.; Liu, X. K.; Wang, Li; Zheng, S. X.; Tam, D.G.
Two of the three types of MICE magnets will ha