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SmD1 Modulates the miRNA Pathway Independently of Its Pre-mRNA Splicing Function
microRNAs (miRNAs) are a class of small regulatory RNAs that fine-tune gene expression by reducing protein output from their target messenger RNAs and are implicated in myriad physiological and pathological processes. miRNAs are generated from long primary transcripts via sequential actions of the Drosha/Pasha and Dicer ribonucleases. Mature miRNAs are incorporated into the miRISC effector complexes that contain AGO family member proteins and serve as specificity determinants to guide miRISCs to their target RNAs. Previous studies suggested that select proteins implicated in the processing of messenger RNAs are required for the miRNA production/function, but the underlying molecular mechanism is not well understood. Here we show that SmD1, an essential protein implicated in the processing of messenger RNAs, directly interacts with both Pasha and primary miRNA transcripts and is required for optimal miRNA production. Furthermore, SmD1 associates with multiple components of the miRNA effector machinery and is required for miRNA function. Finally, our analysis reveals that defects in the miRNA pathway can be uncoupled from those in messenger RNA processing, and that the miRNA biogenesis and messenger RNA processing machineries are physically and functionally distinct entities. Our data thus suggests that SmD1 modulates the miRNA pathway independent of its role in messenger RNA processing.
Published in the journal:
. PLoS Genet 11(8): e32767. doi:10.1371/journal.pgen.1005475
Category: Research Article
doi: 10.1371/journal.pgen.1005475
microRNAs (miRNAs) are a class of small regulatory RNAs that fine-tune gene expression by reducing protein output from their target messenger RNAs and are implicated in myriad physiological and pathological processes. miRNAs are generated from long primary transcripts via sequential actions of the Drosha/Pasha and Dicer ribonucleases. Mature miRNAs are incorporated into the miRISC effector complexes that contain AGO family member proteins and serve as specificity determinants to guide miRISCs to their target RNAs. Previous studies suggested that select proteins implicated in the processing of messenger RNAs are required for the miRNA production/function, but the underlying molecular mechanism is not well understood. Here we show that SmD1, an essential protein implicated in the processing of messenger RNAs, directly interacts with both Pasha and primary miRNA transcripts and is required for optimal miRNA production. Furthermore, SmD1 associates with multiple components of the miRNA effector machinery and is required for miRNA function. Finally, our analysis reveals that defects in the miRNA pathway can be uncoupled from those in messenger RNA processing, and that the miRNA biogenesis and messenger RNA processing machineries are physically and functionally distinct entities. Our data thus suggests that SmD1 modulates the miRNA pathway independent of its role in messenger RNA processing.
miRNAs are a class of ~22–24 nt endogenous regulatory RNAs present in all cell types of multicellular organisms [1,2]. By regulating the expression of diverse target RNAs, miRNAs play a key role in myriad biological processes including development, homeostasis, and innate immunity. In Drosophila, canonical miRNA biogenesis starts with RNA polymerase II-mediated transcription of long stem-loop primary miRNA transcripts (pri-miRNAs). They are processed in the nucleus by the Drosha/Pasha ribonuclease III (RNase III) microprocessor complex into ~60–70 nt precursor miRNAs (pre-miRNAs), via a reaction referred to as cropping [3,4,5]. Precursor miRNAs are subsequently exported to the cytoplasm by Exportin5/Ran-GTP and further processed in a dicing reaction by a second RNase III complex, the Dicer 1 (Dcr-1)/Loqs-PB complex, thereby liberating ~22–24 nt miRNA duplexes [6,7,8,9,10,11,12]. The mature miRNA strand of the duplex is predominantly incorporated into Argonaute 1 (AGO1)-containing miRNA-induced silencing complexes (miRISC). miRISC in turn engages target mRNAs via complementary base pairing between the seed region of miRNAs (positions 2–8) and miRNA-binding sites (primarily in the 3' UTR of target mRNAs), and represses gene expression post-transcriptionally by promoting target mRNA destabilization and/or translation inhibition [13,14,15,16,17,18].
Over a decade of investigation has identified a collection of core components of the miRNA biogenesis and functional machineries, and delineated the framework of the molecular mechanism underlying the miRNA pathway. However, our knowledge of the miRNA pathways is far from complete, and many accessory factors that modulate miRNA biology await identification and functional characterization. A number of recent studies highlight extensive crosstalk between pre-mRNA splicing and small RNA-mediated gene silencing pathways. For example, the core RISC component AGO2 has been implicated in modulating pre-mRNA splicing both in mammals and in Drosophila [19,20]. Conversely, select splicing factors have been shown to impact small RNA-mediated gene silencing pathways. It has been reported that mutations in genes encoding a subset of splicing factors compromise RNAi in the fission yeast Schizosaccharomyces pombe [21], and that in plants, mutations in splicing factor genes compromise small RNA biogenesis [22]. In addition, the multifunctional human RNA-binding protein hnRNP A1, which regulates alternative splicing, impacts the processing of miR-18a and let-7a [23,24]. Furthermore, the KH-type splicing regulatory protein (KSRP) has been shown to positively regulate the biogenesis of miR-155 and let-7 [25,26]. Moreover, genome-wide RNAi screens conducted in C. elegans and cultured Drosophila cells show that depletion of certain splicing factors compromises RNAi [27,28,29]. Finally, a recent analysis of phylogenetic conservation of candidate RNAi factors suggests that select splicing factors are required for small RNA-mediated gene silencing [30].
SmD1, together with six other small ribonucleoprotein particle (snRNP) proteins (SmB, SmD2, SmD3, SmE, SmF and SmG), form a heptameric ring structure surrounding the U-rich small nuclear RNAs (snRNAs) [31]. These snRNP proteins constitute core components of the snRNP and play a key role in pre-mRNA splicing [32]. We recently showed that SmD1 depletion in cultured Drosophila cells compromises small interfering RNA (siRNA) biogenesis and function independently of its role in pre-mRNA splicing [33]. In the current study, we investigate the role of SmD1 in the miRNA pathway. We find that SmD1 depletion leads to a reduction in levels of mature miRNAs, which is accompanied by a derepression of the corresponding target messenger RNAs and a concomitant accumulation of primary miRNA transcripts. In addition, SmD1 associates with the microprocessor component Pasha and is required for optimal microprocessor activity. In contrast, SmD1 is dispensable for Dicer-mediated processing of pre-miRNAs into mature miRNAs. Furthermore, our analysis reveals that SmD1 is required for miRNA function besides its role in miRNA biogenesis. Specifically, SmD1 associates with the miRISC components AGO1 and GW182, and is required for optimal miRISC function. Moreover, we show that select splicing factors such as SmD1, but not pre-mRNA splicing per se, modulate the miRNA pathway, as defects in miRNA biogenesis and in pre-mRNA splicing can be uncoupled, and that SmD1 inactivation does not affect the expression of canonical miRNA pathway components. Finally, PAR-CLIP analysis identifies numerous SmD1-binding events across the transcriptome and reveals direct SmD1-miRNA interactions. Taken together, our study identifies SmD1 as a new modulator of the miRNA pathway at multiple levels, and provides direct evidence to support and extend the notion that select splicing factors are critical modulators of small RNA pathways in complex multicellular organisms, beyond the context of the spliceosome.
SmD1 is required for miRNA biogenesis and/or stability
We first examined small RNA expression profiles in control S2 cells or cells depleted of Drosha, Dcr-2 or SmD1 (Fig 1A and S1 Table). As expected, compared with control samples, depletion of Drosha led to a reduction in the proportion of miRNAs in the total small RNA population. As a consequence, the proportions of all classes of endogenous siRNAs expanded, including those derived from the Flock House Virus (FHV), transposable element (TE), convergently transcribed RNAs (cis-NAT) and hpRNAs. In contrast, depletion of the siRNA biogenesis enzyme Dcr-2 caused the opposite phenotype. Notably, SmD1 knockdown led to a shrinkage of the miRNA population and an artificial expansion of endo-siRNAs. Considering our previous findings showing that SmD1 is required for siRNA biogenesis and SmD1 depletion led to a reduction in levels of endo-siRNAs [33], our data strongly suggest that SmD1 depletion can cause a comparable or even stronger degree of reduction in levels of miRNAs than in levels of siRNAs. To test this directly, we measured levels of endogenous siRNAs and miRNAs by Northern blot in SmD1 knockdown cells. Consistent with our recent finding [33], SmD1 depletion led to a marked decrease in levels of the endogenous siRNA esi-2.1 (Fig 1B and 1E). Importantly, levels of several miRNAs, including miR-33, miR-34, miR-276a, miR-317, miR-2b, miR-184 and miR-bantam, were significantly reduced upon SmD1 knockdown (Fig 1B–1G), reminiscent of the phenotype elicited by the loss of the canonical miRNA biogenesis enzyme Drosha. As a negative control, depleting canonical siRNA pathway components such as Dcr-2 or AGO2 predominantly affected levels of esi-2.1, but not miRNAs (Fig 1B–1E). To rule out potential off-target effects associated with dsRNAs, we tested an independent dsRNA against SmD1 and observed a similar impact on small RNA levels (S1 Fig). We also detected a significant increase in steady-state levels of a subset of target mRNAs for several miRNAs implicated in cell proliferation and apoptosis (Reaper, E2f1 and Socs36E as targets for miR-2b, miR-184 and miR-bantam, respectively) (Fig 1H) [34,35], consistent with the notion that miRNAs repress target gene expression by inhibiting mRNA translation and promoting target mRNA decay. We conclude that SmD1 is required for optimal miRNA biogenesis and/or stability.
Besides the afore-mentioned miRNAs, which are constitutively expressed in S2 cells, we also examined levels of the miRNA let-7, which is not expressed in naïve S2 cells but becomes highly induced upon treatment with 20-hydroxyecdysone (20-E) [36]. We detected lower levels of let-7 in SmD1-depleted cells (S2 Fig). These data reinforce the notion that SmD1 is required for optimal miRNA biogenesis. let-7 is highly expressed in the Drosophila heart, and is required for Drosophila heart function. Interestingly, depletion of SmD1 specifically in cardiac cells of adult Drosophila causes several defects in cardiac function (S3 Fig). These observations suggest that the cardiac phenotype elicited by SmD1 depletion is linked, at least in part, to defects in let-7 biogenesis.
SmD1 is selectively required for the processing of pri-miRNAs into pre-miRNAs
miRNA biogenesis consists of multiple steps, including Drosha-mediated processing of pri-miRNAs into pre-miRNAs and Dcr-1-mediated conversion of pre-miRNAs into mature miRNAs, referred to as cropping and dicing, respectively (Fig 2A). To further define the biochemical step(s) of miRNA biogenesis that require SmD1, we examined the impact of SmD1 inactivation on levels of pri-miRNAs and pre-miRNAs. As expected, we detected a marked accumulation of several pri-miRNAs in S2 cells depleted of the microprocessor component Drosha (S4 Fig). Importantly, SmD1 phenocopies Drosha, albeit to a moderate extent (Fig 2B), suggesting that SmD1 is required for microprocessor-mediated processing of pri-miRNAs. To assess whether SmD1 is required for Dcr-1-mediated processing of pre-miRNAs into mature miRNAs, we treated S2 cells with dsRNAs targeting the firefly luciferase (as control) or SmD1, together with dsRNAs targeting Dcr-1 (to facilitate the detection of pre-miRNAs), and performed Northern blot to measure levels of pre-miRNAs. We found that compared to control samples, loss of SmD1 caused a reduction in levels of pre-miR-184 (Fig 2C). These observations strongly suggest that SmD1 is dispensable for Dcr-1-mediated processing of pre-miRNAs into mature miRNAs, as otherwise we would have detected an accumulation of pre-miRNAs upon SmD1 inactivation. We note that it remains formally possible that SmD1 is required for the processing of pre-miRNAs into mature miRNAs, and the observed decrease in the overall pre-miRNA levels upon SmD1 knockdown is an accumulative effect of impaired microprocessor activity (leading to less pre-miRNA production) and less efficient Dcr-1-mediated pre-miRNA processing (leading to accumulation of pre-miRNAs). Next, to definitively elucidate the requirement for SmD1 in different steps of miRNA biogenesis, we prepared total or cytoplasmic lysates from S2 cells treated with various dsRNAs, incubated lysates with radiolabeled pri-miRNAs or pre-miRNAs, and measured the microprocessor or Dcr-1 activities by monitoring the production of pre-miRNAs or mature miRNAs, respectively. Our analysis revealed that SmD1-deficient cell lysate is as competent as the control lysate in carrying out Dcr-1-mediated processing of pre-miRNAs into mature miRNAs (Fig 2D). In contrast, SmD1-deficient cell lysate displays significant defects in Drosha-mediated conversion of pri-miRNAs into pre-miRNAs (Figs 2E–2G and S5). These data demonstrate that SmD1 is specifically required for cropping, but is dispensable not the dicing step during miRNA biogenesis.
SmD1 interacts with the microprocessor component Pasha
Having shown that SmD1 is required for optimal microprocessor activity, we next examined whether SmD1 associates with components of the microprocessor. Consistent with the requirement for SmD1 in the cropping step, we found that at Flag-tagged SmD1, but not the control protein Ran, co-immunoprecipitated with the endogenous microprocessor component Pasha (Fig 3A). In addition, the recovery of Pasha in the SmD1 complex is resistant to RNase A treatment, indicating RNA-independent protein-protein interactions. It remains to be determined whether the observed SmD1-Pasha interaction is direct or through protein intermediates. While we were unable to detect endogenous Drosha in the SmD1 complex by immunoblotting, most likely due to the low sensitivity of the Drosha antibody, moderate but clearly above background levels of microprocessor activity can be recovered from immunopurified endogenous SmD1 complex (Fig 3B). These observations underscore the functional relevance of the observed SmD1-Pasha interaction. Next, we performed RNA immunoprecipitation (RIP) assays to assess whether SmD1 could associate with pri-miRNAs, the substrates for the microprocessor. Our analysis revealed significant enrichment in the SmD1 complex all six pri-miRNAs that we have examined, the degree of which matched or even exceeded that of cognate SmD1-binding small nuclear RNAs (Fig 3C). In contrast, no significant enrichment of the control mRNA rp49 was observed. The observed interactions of SmD1 with Pasha and pri-miRNAs suggest that SmD1 might modulate miRNA biogenesis by serving as a molecular bridge to facilitate the recognition of pri-miRNAs by the microprocessor. To test this possibility, we performed RIP assay using a stable cell line expressing TAP-tagged Pasha and detected a significant enrichment of pri-miRNAs in immunopurified TAP-Pasha complex (S6 Fig). Importantly, recovery of pri-miRNAs in the Pasha complex was severely blunted upon depletion of SmD1 (Fig 3D). Interestingly, we also observed a moderate degree of enrichment of the control mRNA rp49 in the Pasha complex. This is consistent with the notion that the microprocessor complex potentially associates with and regulates the expression of a number of cellular mRNAs (S6 Fig) [37,38]. However, the association of the rp49 mRNA with Pasha seems to be largely unaffected by SmD1 depletion (Figs S6 and 3D).
To investigate the molecular detail of the SmD1-Pasha interaction, we next sought to map the protein domains responsible for this interaction. We generated a series of T7-tagged full length and truncated Pasha proteins and examined their capability of interacting with Drosha and SmD1 by performing co-immunoprecipitation assays. As expected, we found that full length Pasha was able to co-immunoprecipitate with endogenous Drosha in cultured Drosophila S2 cells (Fig 3E, lane 7). In addition, the C-terminal fragment of Pasha (334–642) is also capable of pulling down Drosha (Fig 3E, lane 10). Our data are consistent with previous reports showing that the C-terminal fragment of DGCR8 interacts with Drosha in mammals [39,40]. Note that T7-tagged Pasha127–642 was unable to co-immunoprecipitate with endogenous Drosha, possibly due to inefficient folding and/or presentation of the T7 epitope in native T7-Pasha127–642 protein in total cell lysate, as T7-Pasha127–642 was poorly recovered in the anti-T7 immunoprecipitate (Fig 3E, lane 11, lower panel). In contrast, T7-Pasha127–642 was readily detectable in input samples, most likely because the T7 epitope can be efficiently recognized by the anti-T7 antibody in denatured T7-Pasha127–642 (Fig 3E, lane 5, lower panel). Next, we examined whether various truncated Pasha proteins can co-immunoprecipitate with SmD1. We found that Flag-tagged SmD1 was able to pull down both full length and a number of truncated Pasha mutants containing the region spanning amino acids 127–333 (Fig 3F, lanes 7–9, 11). Thus, it appears that distinct regions of Pasha are required for the Pasha-Drosha and Pasha-SmD1 interactions (Fig 3G). The stoichiometry of the SmD1-microprocessor complex is currently not clear, and it remains to be determined whether the observed Drosha-Pasha and SmD1-Pasha interactions are mutually exclusive, or all three proteins are present in the same complex. Collectively, these data demonstrate that SmD1 associates with both components of the miRNA biogenesis machinery and primary miRNA transcripts, and that SmD1 is required for optimal recognition of the pri-miRNAs by the microprocessor.
SmD1 is required for miRNA function
Several lines of evidence point to a possible role of SmD1 in the effector phase of the miRNA pathway (i.e., miRISC assembly and function) besides its involvement in miRNA biogenesis: 1) SmD1 modulates siRISC assembly and function, and associates with several siRISC components, including AGO2 [33]; 2) SmD1, but not the control protein Ran, co-immunoprecipitates with miR-2b [33]; 3) in mammals SNRPD1 (ortholog of Drosophila SmD1) and AGO2 (a canonical miRISC component) interact with each other [33]; and 4) a considerable fraction of SmD1 is present in the cytoplasm (Fig 4A), where miRISC assembly and function primarily take place. To examine this possibility, we transfected SmD1-depleted S2 cells with a synthetic let-7 miRNA duplex together with a Renilla luciferase reporter construct carrying 8 copies of imperfect let-7-binding sites in the 3' UTR. A firefly luciferase reporter lacking miRNA-binding sites serves as control. Defects in miRISC assembly/function are expected to be reflected as an increase (de-repression) in reporter activity compared to the negative control. It is worth noting that employing a synthetic mature miRNA duplex in the assay effectively circumvents the confounding factor that SmD1 is required for miRNA biogenesis. In addition, we chose let-7 because of its extremely low basal expression in S2 cells, thereby reducing background. Our analysis revealed that compared to control knockdown cells, SmD1-depleted cells display a marked de-repression of the let-7 reporter, resembling the phenotype elicited by depletion of the core miRISC component AGO1 (Fig 4B). These data indicate that SmD1 is required for miRNA function. Consistent with this notion, we found that Flag-tagged AGO1, but not the control protein Ran, is capable of co-immunoprecipitating with endogenous SmD1 (Fig 4C). Furthermore, endogenous AGO1 as well as GW182, another component of the miRISC, were detected in SmD1 complex, but not in the control Ran complex or the control immunoprecipitates using a non-immune serum (Fig 4D and 4E). It appears that the SmD1-AGO1 and SmD1-GW182 interactions are not strongly affected by RNase treatment (Fig 4D and 4E). These data demonstrate the interaction between SmD1 and components of the miRISC.
Next, to determine the functional relevance of the SmD1-AGO1 interaction and to directly assess the role of SmD1 in miRISC assembly/function, we examined whether SmD1 depletion impairs the function of miRISC by measuring the slicer activity of AGO1-miRISC programmed by the let-7 miRNA duplex. To circumvent the confounding factor that AGO2 is a much more robust slicer than AGO1 and thus could mask the weak slicer activity of AGO1, we first established stably transfected S2 cells expressing TAP-tagged AGO1. Then we immunopurified and immobilized the AGO1 complex onto agarose beads and incubated the AGO1 complex with cytoplasmic lysates (from either control cells or SmD1 knockdown cells) together with the let-7 miRNA duplex. The beads were subsequently washed thoroughly and the slicer activity of the bead-bound let-7-AGO1 miRISC against an mRNA substrate carrying a perfect let-7 binding site was measured. This assay revealed a significantly weaker slicer activity of the AGO1-miRISC assembled in SmD1-depleted cell lysate than that assembled in control lysates (Fig 4F and 4G), suggesting that SmD1 is required for efficient miRISC assembly and/or function. To examine whether SmD1 is required for the loading of miRNAs into AGO1, which is the first step of miRISC assembly, we depleted SmD1 in TAP-AGO1-expressing S2 cells, and measured the levels of endogenous miRNAs in immuno-purified TAP-AGO1 complex (Fig 4H). Note that as a control for the amount of TAP-AGO1 expressed from the transgene across different samples, we used identical amount of cell lysates in each immunoprecipitation. Compared with controls, the absolute levels of both miR-2b and miR-184 were markedly reduced in AGO1 miRISC recovered from SmD1-depleted cells (Fig 4H and 4I). This is in part due to lower levels of miRNAs in input samples from SmD1 knockdown cells (Fig 4H, compare the input samples). In addition, we calculated AGO1 loading index by normalizing levels of AGO1-bound miRNAs against those in the input. This analysis revealed a moderate decrease in AGO1 loading index upon SmD1 knockdown, suggesting that SmD1 is required for efficient loading of miRNAs into miRISC (Fig 4J). Taken together, these data demonstrate that SmD1 is required for efficient miRISC assembly/function besides its role in miRNA biogenesis.
Transcriptome-wide identification of SmD1-associated RNAs
To comprehensively identify direct SmD1-RNA interaction events across the transcriptome, we optimized the PAR-CLIP protocol in Drosophila S2 cells, recovered and deeply sequenced SmD1-bound RNAs (S7A and S7B Fig) [41,42]. Mapped reads are derived from various classes of RNAs (S7C Fig). Our initial analysis identified 2180 SmD1 binding sites (clusters) across the transcriptome. Of these, 1729 unique peaks have passed non-adaptive filtration (see Materials and Methods) and were used for subsequent analyses (S2 Table). Among them, 96 map to unannotated genomic regions. For the remaining 1633 clusters, 1553 (95%) and 80 (5%), respectively, map to the annotated coding and non-coding RNAs (Figs 5A and S8; S3 and S4 Tables). As expected, snRNAs, the cognate binding partners for SmD1, were abundantly present in the dataset, so were sequences derived from the endogenous siRNA esi-2.1 precursor CG4068, consistent with our previous report (Fig 5B and S2 Table) [33]. In addition, SmD1 binding events in the vicinity of splice junctions were found (S5 Table), thereby providing supporting evidence for well-documented role of SmD1 in regulation of pre-messenger RNA processing. Interestingly, we also identified a substantial number (24) of SmD1-interacting snoRNAs. Importantly, sequences derived from several pri-miRNAs, including Bantam, miR-2 family, miR-11, miR-33 and miR-34 were also recovered, thus demonstrating direct interactions between SmD1 and pri-miRNAs (Figs 3C, 5C, 5D and S9; S6 Table). Interestingly, among the primary miRNA transcripts that we have examined so far, miR-33 and miR-34 are the top two highly enriched in immunopurified SmD1 complex (Figs 3C, 5C and 5D). Notably, a major SmD1 binding peak directly overlaps with mature miR-34 (Fig 5D), suggesting that SmD1 may directly associate with mature miRNAs (in the context of miRISC). This is consistent with our previous findings that immunopurified SmD1 complex contains mature miRNAs [33]. An alternative and non-mutually exclusive possibility is that SmD1 could bind the sequence segment corresponding to mature miR-34 within the primary miRNA transcript.
Loss of SmD1 does not functionally alter the expression of canonical miRNA factors
It is conceivable that SmD1 depletion indirectly impacts the miRNA pathway by modulating the expression of genes encoding canonical miRNA factors. We previously performed pair-end RNA-sequencing in both control cells and cells depleted of SmD1 [33]. Consistent with the role of SmD1 in pre-mRNA splicing, our analysis revealed that the splicing pattern of ~25% cellular mRNAs is altered in SmD1-depleted cells. Importantly, we found no significant changes in the mRNA levels of canonical miRNA factors except an increase in levels of Drosha and a decrease in loqs-RD, whose encoded product is dispensable for the miRNA pathway (S10 Fig). Most importantly, immunoblotting assays revealed no significant changes in protein levels of canonical miRNA factors in SmD1-depleted cells except for an increase in levels of Loqs-PB and a concomitant reduction in Loqs-PD (Fig 6A) [33]. Given our findings that SmD1 impacts the cropping and miRISC assembly/function (Figs 2E–2G, 4 and S5), which do not require Loqs proteins, it is unlikely that the observed changes in levels of Loqs protein isoforms could account for the miRNA biogenesis and function defects elicited by SmD1 depletion. We conclude that SmD1 depletion does not functionally impact the expression of genes encoding canonical components of the miRNA pathway.
Select splicing factors, but not splicing per se, modulate the miRNA pathway
To determine if a broader link exists between the miRNA pathway and splicing, we examined additional snRNP (Sm) proteins for their potential involvement in the miRNA pathway. We found that depletion of several snRNP proteins led a reduction in levels of miR-2b, reminiscent of the SmD1 knockdown phenotype (Fig 6B). In contrast, levels of miR-2b remained unchanged in SmF knockdown cells. Furthermore, SmF knockdown did not impact in microprocessor activity either, even though SmF-depleted cells displayed obvious alteration in the splicing pattern of the CG13887 pre-mRNA, to the same extent as that observed in SmD1-depleted cells (Figs S11, 6C and 6D). These data clearly show that the miRNA defects elicited by SmD1 depletion can be uncoupled from pre-mRNA splicing defects, as the miRNA pathway appears to be intact in SmF-depleted cells, even though these cells display profound defects in pre-mRNA splicing.
To further dissect the relationship between the miRNA and pre-mRNA splicing machineries, we examined whether additional snRNP proteins are capable of interacting with Pasha. We found that besides SmD1, SmD2 is also capable of co-immunoprecipitating with Pasha (Fig 6E). In contrast, no interaction can be detected between Pasha and SmB or SmF, even though both SmB and SmF are capable of pulling down endogenous SmD1, most likely the SmD1 fraction in the spliceosome (Fig 6E). Furthermore, upon examining levels of pri-miRNA and snRNA species in various immuno-purified Sm protein complexes, we found that SmD1 consistently outperforms SmF in binding to various pri-miRNAs. In contrast, we detected significantly higher levels of the U4 snRNA in the SmF complex compared to those present in the SmD1 complex (Fig 6F). We note that Flag-tagged Sm proteins were employed in these assays and that these exogenous proteins have to complete with their endogenous counterparts for binding to their RNA partners. However, since we are measuring levels of various RNA cargos in the same sample, it allows us to make a direct comparison regarding the relative affinity between various Sm proteins and their RNA partners. These data suggest that compared with SmF, SmD1 appears to display a more prominent role in miRNA biogenesis, whereas SmF seems to be more dedicated to pre-mRNA splicing. In addition, these data also indicate that the spliceosome and miRISC are functionally distinct entities. To address this further, we immunopurified the microprocessor or the snRNP complexes by TAP-Pasha IP or SmD1 IP, respectively, and examined the presence of various snRNAs and pri-miRNAs in these complexes. As expected, snRNAs were highly enriched in the SmD1 complex (Fig 3C). In contrast, snRNAs were largely absent in the microprocessor (Fig 6G). On the other hand, several primary miRNA transcripts were highly enriched in the microprocessor (Figs 3D and S6). Taken together, these data demonstrate that select splicing factors, but not splicing per se, influence the miRNA pathway, and that the miRNA biogenesis machinery and the spliceosome are physically and functionally distinct.
Accumulating evidence suggests extensive crosstalk between pre-mRNA splicing and small RNA-mediated gene silencing pathways. For example, the core component of the RNAi effector machinery AGO2 plays a key role in the regulation of pre-mRNA splicing [19,20]. Conversely, a number of splicing factors have been implicated in modulating small RNA-mediated gene silencing [21,22,23,24,25,26,27,28,29,30]. We previously showed that the core snRNP splicing factor SmD1 is required for optimal biogenesis and function of siRNAs [33]. In the current study we report that SmD1 also plays a key role in modulating the miRNA pathway. Specifically, SmD1 interacts with the microprocessor component Pasha and with primary miRNA transcripts, and is selectively required for efficient recognition of primary miRNA transcripts by the microprocessor and the conversion of pri-miRNAs into pre-miRNAs. In addition, SmD1 associates with the miRISC components AGO1 and GW182, and is required for miRISC assembly/function. We further show that only select splicing factors, but not pre-mRNA splicing per se, modulate the miRNA pathway, since defects in pre-mRNA splicing can be uncoupled from defects in the miRNA pathway, and that the molecular machineries executing pre-mRNA splicing and miRNA biogenesis and function are physically and functionally distinct entities.
Our analysis reveals that SmD1, but not a closely related snRNP protein SmF, is required for optimal miRNA biogenesis. These observations indicate that SmD1 belongs to a select group of splicing factors that modulate small RNA pathways beyond the context of the splicing machinery. SmD1 associates with the N-terminus of Pasha, whereas the C-terminal domain of Pasha is sufficient to interact with Drosha. It is currently unclear whether the SmD1-Pasha and Drosha-Pasha interactions take place in a mutually exclusive manner, or alternatively there exists a Drosha-Pasha-SmD1 complex. Our observation that the immunopurified SmD1 complex is capable of carrying out the conversion of pri-miRNAs into pre-miRNAs lends support to the latter possibility (Fig 3B). Of note, several splicing factors have been shown to co-purify with the microprocessor [3]. Our study, together with a recent report that implicates FUS in miRNA biogenesis [43], demonstrates that select splicing factors are functionally associated with the microprocessor in higher multicellular organisms. While it is possible that SmD1 couples splicing and processing of pri-miRNA transcripts by recruiting the microprocessor to nascent pri-miRNA transcripts that undergo co-transcriptional splicing, our observations that depletion of SmF, a related snRNP splicing factor, does not impact miRNA biogenesis (Figs 6B and S11), that neither SmB nor SmF is able to co-immunoprecipitate with Pasha (Fig 6E), and that immunopurified SmD1 and Pasha complexes harbor overlapping yet distinct sets of cargo RNAs (Figs 3C, 6F and 6G), argue against this possibility.
While SmD1 is clearly required for miRNA biogenesis, it may operate in the context of multimeric protein complexes to execute this function. For example, our data show that SmD2 is as competent as SmD1 in pulling down primary miRNA transcripts and Pasha, and depletion of SmD2 led to a reduction in both miRNA levels and microprocessor activity (Figs 6B, 6E, 6F and S11). Together with previous studies that report the presence stable SmD1-SmD2 subcomplex, [31,44], our data raise the possibility that the SmD1/D2 sub-assembly may execute a moonlighting function during miRNA biogenesis beyond the context of the spliceosome. We found that adding back purified recombinant SmD1 or lysates from cells over-expressing SmD1 was not sufficient to restore the microprocessor activity or miRISC assembly/function in SmD1-depleted cell lysate (S12 and S13 Figs). These observations suggest that additional SmD1 co-factor(s) (such as SmD2) in the context of multimeric complexes of appropriate stoichiometry may be necessary to functionally impact the miRNA pathway. An alternative possibility is that SmD1 depletion leads to altered expression of unknown protein(s) involved in miRNA biology, which underlies the defects in the miRNA pathway elicited by SmD1 knockdown. However, our findings lend strong support to the former scenario.
Interestingly, our data uncover a differential dedication of various Sm proteins to the miRNA pathway and pre-mRNA splicing. For example, SmD1 and SmD2 consistently outperform SmF in binding to various pri-miRNAs, whereas the U4 snRNA is significantly more enriched in the SmF complex than in the SmD1 complex. (Fig 6F) This is consistent with our findings showing that SmD1 and SmD2, but not SmF, is required for the miRNA pathway. These observations also indicate the presence of sub-spliceosomal assemblies or novel SmD1-containing complexes that impact the miRNA pathway. Further supporting this notion, our co-immunoprecipitation assay reveals that Flag-tagged SmD1 is capable of pulling down endogenous SmD1 (Fig 6E, lane 8). Most likely this interaction takes place beyond the context of the spliceosome, as the snRNP ring structure contains only a single copy of each Sm protein. Interestingly, depleting either SmB or SmD2, immediate neighbors to SmD1 in the snRNP ring structure, led to a reduction in miRNA levels and microprocessor activity. However, only SmD2, but not SmB, is capable of co-immunoprecipitating with Pasha (Fig 6E). Identification of the complete collection of microprocessor-associated splicing factors and unraveling the stoichiometry of the microprocessor/SmD1-containing complexes should provide insights into the function of microprocessor-associated splicing factors in miRNA biogenesis.
Besides its involvement in the initiation phase of the miRNA pathway (miRNA biogenesis), SmD1 is also required for the effector phase of the miRNA pathway, as SmD1 depleted cells display defective miRISC assembly/function. This is manifested in part by defects in the loading of miRNAs into AGO1-miRISC (Fig 4H–4J). It remains to be determined whether SmD1 is similarly required for additional steps of miRISC assembly, including miRNA duplex unwinding and miRNA star strand removal.
We report here that the Drosophila SmD1 associates with the microprocessor and impacts the cropping step of miRNA biogenesis (Fig 6H). Interestingly, in C. elegans the SmD1 ortholog SNR-3 co-purifies with Dcr-1 [45]. It would be informative to address whether SNR-3 similarly impacts miRNA biogenesis in worms. In addition, we show here that SmD1 associates with the core miRISC component AGO1 in flies and impacts miRNA function (Fig 6H). An analogous observation has been made regarding the role of SmD1 in the siRNA pathway, where it associates with the siRISC component AGO2 and is required for siRISC assembly/function. Furthermore, we report previously that the human orthologs of SmD1 and AGO2 associate with each other [33]. These findings raise the possibility that the role of SmD1 in modulating small regulatory RNA biogenesis and Argonaute-RISC assembly and function may be evolutionarily conserved.
Materials and Methods
Cell culture, transfection, and RNAi
Drosophila S2 and S2-NP cells were maintained in Schneider's medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen). S2-NP cells stably expressing Flag-SmD1 were generated by transfection with pRmHa-3-Flag-SmD1 and the selection marker plasmid pHS-neo using the calcium phosphate method, followed by selection in medium containing 400 μg/mL G418 (Calbiochem). dsRNA treatment was performed as described previously [27,46]. Briefly, ~2 × 106 S2 or S2-NP cells were seeded in 6-well plates for 24 h and then transfected with 3 μg of the appropriate dsRNA. Two days later, the cells were harvested, replated in 6-cm plates for 24 h, and then treated again with 9 μg dsRNA. Three days later, the cells were harvested and used in assays. For Renilla luciferase reporter assays, transfections were performed in a 384-well format using HiPerFect (Qiagen).
DNA constructs and antibodies
DNA fragments encompassing the coding regions of SmD1, SmB, SmD2, SmF, Drosha, AGO1, and full length and truncated Pasha, together with Flag, T7 or TAP epitope tags were amplified by PCR and cloned into pRmHa-3 or pMK33-NTAP vectors. Anti-SmD1 antibody was generated by immunizing rabbits with a synthetic peptide (ProSci Inc.).
Protein/RNA immunoprecipitation and immunoblotting
Cells were lysed in lysis buffer (20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM DTT, 1 mM orthovanadate) supplemented with protease inhibitor cocktail (Roche). Cleared total lysates were immunoprecipitated with antibodies against Flag (Sigma). Both input and immunoprecipitated samples were analyzed by SDS-PAGE followed by immunoblotting with antibodies against Flag (Sigma), T7 (Novagen), AGO1 (Abcam), Pasha [5] or GW182 [47]. Other antibodies employed in this study include Drosha and Dcr-1 (gift from Dr. Greg Hannon), Loqs [48] or Tubulin (Sigma), as indicated. RNase treatment of the immunoprecipitates was performed as previously described [27,46]. To detect protein-RNA interactions, RNA was extracted from the immunoprecipitates by treatment with TRIzol and subject to RT-qPCR analysis using gene-specific primers.
Northern blotting
Northern blotting was performed as previously described [46,48]. In brief, total cellular or co-immunoprecipitated RNA was isolated with TRIzol (Invitrogen). Samples of 15 μg RNA were separated on 15% denaturing polyacrylamide gels and transferred to Hybond-N+ membranes (Amersham Biosciences) in 1X TBE buffer. Small RNAs were UV crosslinked to the membranes, and the membranes were prehybridized in hybridization buffer for 2 h. DNA probes complementary to the appropriate strands were 5′ radiolabeled and incubated with membranes overnight at 37°C. Membranes were washed twice in 1X SSC with 0.1% SDS at 42°C, and then exposed to Phosphorimager screens for 12–48 h. Membranes were stripped by the addition of boiling 0.1% SDS solution and incubated for 30 min.
Cropping, dicing and slicing assays
Cropping, dicing and slicing assays were performed as previously described, with minor modifications [49,50]. Briefly, for the cropping assay, total S2-NP cell lysates were prepared by sonicating cell suspension in lysis buffer (30 mM HEPES-KOH, pH 7.0, 100 mM potassium acetate, 2 mM magnesium acetate, 5 mM DTT, 20% glycerol, and 1X EDTA-free protease inhibitors (Roche)) for 5 times (5 sec each with 2 min interval at 30% duty cycle). Primary miRNAs were synthesized using a T7 MEGAscript in vitro transcription kit (Ambion) with α-32P-GTP, and gel purified. Aliquots of 10 μl of cell lysates containing the same amount of total protein were incubated in a final volume of 20 μl reaction mixture (30 mM HEPES-KOH, pH 7.0, 100 mM potassium acetate, 2 mM magnesium acetate, 5 mM DTT, 10% glycerol, 1 mM ATP, 10 mM creatine phosphate, 0.06 U/μl creatine kinase (Roche), 0.1 U/μl ribonuclease inhibitor (Promega), 10 ng/μl yeast tRNA, and 2000–10,000 cpm pri-miRNA substrate) at 25°C for 2.5 h.
For the dicing assay, cytoplasmic extracts from frozen S2-NP cells were prepared by thawing cells in a hypotonic buffer composed of 10 mM HEPES-KOH (pH 7.0), 2 mM magnesium acetate, 0.1% β-mercaptoethanol, and 1X EDTA-free protease inhibitors (Roche). Radiolabeled pre-miRNA substrate was prepared by incubating the pre-let-7 synthetic RNA oligo with T4 polynucleotide kinase (New England Biolabs) and γ-32P-ATP and subsequently gel-purified. Aliquots of 6 μl of cell lysates containing the same amount of total protein were incubated in a final volume of 10 μl reaction mixture (20 mM HEPES-KOH, pH 7.0, 2 mM DTT, 2 mM magnesium chloride, 1 mM ATP, 25 mM creatine phosphate, 0.06 U/μl creatine kinase (Roche), 0.8 U/μl ribonuclease inhibitor (Promega), and 2000–10,000 cpm pre-let-7 substrate) at 25°C for 1 h.
For the slicing assay, the capped Renilla luciferase mRNA substrate containing 1 copy of perfect let-7 binding site in the 3' UTR was synthesized using a MEGAscript T7 in vitro transcription kit, incubated with Vaccinia virus capping enzyme (New England Biolabs) and α-32P-GTP, and gel purified. Minimal AGO1-miRISC was prepared by incubating TAP-AGO1 cell lysates with IgG beads with gentle rocking at 4°C overnight. The beads were thoroughly washed in hypotonic buffer and incubated with cytoplasmic lysates from either SmD1 knockdown or control cells in a final volume of 50 μl reaction mixture (8 mM HEPES-KOH (pH 7.0), 60 mM potassium acetate, 5 mM DTT, 1 mM ATP, 25 mM creatine phosphate, 0.03 U/μl creatine kinase, 0.2 U/μl ribonuclease inhibitor, and 1 mM let-7 miRNA) at 25°C for 30 min. The beads were subsequently thoroughly washed in hypotonic buffer and incubated in a final volume of 50 μl reaction mixture (8 mM HEPES-KOH (pH 7.0), 60 mM potassium acetate, 5 mM DTT, 1 mM ATP, 25 mM creatine phosphate, 0.03 U/μl creatine kinase, 0.2 U/μl ribonuclease inhibitor, 10 ng/μl yeast tRNA, and 2000–10,000 cpm cap-labeled mRNA substrate) at 25°C for 2 h. After the final incubation step, the cropping, dicing or slicing reaction mixtures were then added to 200 μl proteinase K buffer (200 mM Tris-HCl (pH 7.5), 25 mM EDTA, 300 mM sodium chloride, 2% w/v SDS, and 50 μg/mL proteinase K), incubated at 65°C for 30 min, and extracted with phenol/chloroform (1:1). RNA was precipitated from the supernatant and resolved by 6% (for cropping and slicing) or 15% urea-PAGE (for dicing).
PAR-CLIP and library construction and RNA-Seq data analysis
PAR-CLIP procedure and library construction were conducted as previously reported [41,42]. Sequenced reads were trimmed of 3' and 5' adaptors (3' = "TGGAATTCTCGGGTGCCAAGG"; 5' = "ATCTCGTATGCCGTCTTCTGCTTG") using flexbar package [51]. Reads that were less than 15 nucleotides in length were discarded. In order to accurately analyze each binding event without the confounding bias introduced by repetitive regions, we identified and removed reads that align to rRNA, tRNA and RepeatMasker sequences (UCSC BDGP R5/dm3 and FlyBase FB2014_03). Elimination of reads mapped to repetitive sequences has significantly affected the overall read yield available for further analyses, thus complicating discovery of SmD1 binding sites. The remaining reads were aligned to Drosophila genome with bowtie algorithm [52]. Mapped locations were only reported for the optimal mismatch-stratum for each read up to a maximum of ten different locations. The resulting alignment was processed with PARALYZER tool (v1.1) [53]. All clusters that have two or more T to C conversion locations were reported. The discovered clusters were further filtered to exclude those where a) read coverage was lower than 10, b) ModeScore< = 0.6, and c) number of unique locations having at least one conversion event exceeded 2. The above non-adaptive filtration helped to remove potential false positive binding events in low coverage regions, where PARALYZER's signal-to-noise estimation becomes less reliable. The location that a cluster mapped to, relative to a known transcript, was determined based on the FlyBase genome annotation (release 5.57). To assess the extent of SmD1 binding in miRNA precursor regions, ±10Kb around the known miRNA locus were examined and overlapping read clusters detected with BEDTools suite. To evaluate the role of SmD1 on regulation of mRNA splicing, coordinates of exon junctions annotated in FlyBase were extracted. Next, regions of interest around exon–intron junctions at the 5′ and 3′ ends of introns were determined. These loci included: a) 15 bp region around 5' splice site, of which 5bp were inside the intron, and b) 28bp region around 3' splice site, where only 3bp overlapped with the 3' exon. Finally, the intersection between SmD1 binding and mRNA splice sites was computed.
Small RNA library construction and data analysis
Small RNA libraries were constructed from gel purified 19–24 nt RNA samples using the TruSeq small RNA sample kit according to manufacturer's manual (Illumina), and sequenced on a GA-II machine. Reads were trimmed of 3' and 5' adaptors (3' = "TGGAATTCTCGGGTGCCAAGG"; 5' = "ATCTCGTATGCCGTCTTCTGCTTG") with cutadapt package (http://dx.doi.org/10.14806/ej.17.1.200) and mapped to the fly genome (rel. 5.57) with Bowtie software suite (v.1.1.1) [52] in a sequential manner {e.g. progressively relaxing constraints for permissive number of mismatches until the limit of 2 was reached (–v[0–2]-best)}. The alignment was processed with HTSeq [54] and read abundances assigned to various genomic elements calculated. To assess read numbers mapping to transposable elements loci we used only LINE-like and LTR elements annotated in RepeatMasker (UCSC BDGP R5/dm3 and FlyBase FB2014_03). Annotation and genomic coordinates for 3p-CIS-NAT, esiRNA loci used to estimate siRNA abundances were obtained from Eric Lai (personal communication). Reads that failed to map to the fly genome were aligned to the flock house virus (FHV) genome and counted.
Data availability
The sequencing results will be submitted to the SRA/ENA repository.
See S7 Table.
Adult fly heart analysis
SmD1 was knocked down in the Drosophila heart by cardiac-specific RNAi using the UAS/Gal4 system [55]. Female tinCΔ4-Gal4 flies [56] were crossed to UAS-shSmD1 (Bloomington stock 34834) and UAS-shGFP (gift from Dr. Norbert Perrimon) respectively. The female F1 progeny was aged for 3 weeks at 25°C and heart function was analyzed using high-speed recordings of semi-dissected hearts [57].
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HTML5 - Basics to Advanced
- 3.5 hours on-demand video
- 2 downloadable resources
- Full lifetime access
- Access on mobile and TV
- Certificate of Completion
Get your team access to 4,000+ top Udemy courses anytime, anywhere.Try Udemy for Business
- Learn to create simple web pages using HTML5
- Learn the basics tags of HTML
- To create Tables in HTML
- To create customizable forms and add form validation
- To include multimedia(audio,video, images) in your web page
- To create your own vector graphics using SVG
- 4 projects to test your knowledge
Hey guys, in this course, we will learn all about HTML5.
We will start from basic topics like adding headings, paragraphs, images, etc and will move towards more advanced topics like adding forms, vector graphics, multimedia and so on.
Hope you are excited to start your journey to become a Web Developer!!
Hey guys, in this video we will explore different code editor options. We will talk about - Notepad, Codepen.io and Visual Studio Code.
Hey guys,
These are the details for the Forms Practice Problem 1:
Image Links:
Google Logo: http://pluspng.com/img-png/google-logo-png-open-2000.png
Facebook Logo: https://cdn4.iconfinder.com/data/icons/social-media-icons-the-circle-set/48/facebook_circle-512.png
Github Logo: https://image.flaticon.com/icons/svg/25/25231.svg
LinkedIn Logo: https://cdn4.iconfinder.com/data/icons/social-media-icons-the-circle-set/48/linkedin_circle-512.png
Background Color Code: #f2f6ff
Border Color: #ccc
Border Width: 1px
Form Heading:
Font Size: 32px
Padding-Top: 56px
Padding-Bottom: 32px
Social Icons:
Width: 50px
Height: 50px
Padding Top and Bottom: 16px
Padding Left and Right: 32px
Input Fields Container:
Padding Top: 80px
Padding Bottom: 40px
Padding Left and Right: 80px
Input Box:
Font Size: 18px
Padding Bottom: 12px
Margin Bottom: 36px
Border Top, Left, Right: 0
Border Bottom: 1px solid #ccc
Submit Button:
Background Color: #0056ff
Padding Top and Bottom: 12px
Padding Left and Right: 24px
Font Size: 18px
Font Color: white
Border: None
Have Fun !!
- None. This course is for absolute beginners.
****UPDATE [06-July-2020]: New Content Added!!!!!****
Around 20 NEW videos added
New Sections on:
Embedding Multimedia
Vector Graphics
And, a Final Project!
Are you a beginner? Do you want to learn web development but do not have any idea where to start?
If yes, then this is THE perfect course for you. In this course, you will learn all the concepts of HTML5, starting from its history to its modern structure. We will cover all the tags which are most widely used in the world of web development. Simultaneously, we will show you some of the best practices of code-writing in HTML5.
HTML is short for 'HyperText Markup Language', which means it is only used to create a structure of the web page. But without some styling, no assignment or project will look good. Thus, we will cover some portion of the CSS required to create simple web pages.
This course will be beneficial for all who are looking to start their career in the web development field and aspire to become a Frontend or Full-stack web developer. This course will be the first step towards your goal to become a web developer. To continue this journey, we would recommend you complete the other languages and skills like CSS, Javascript, ES6, React.
Check out our profile to find related courses that are designed just like this.
PS: More lectures will be uploaded soon.
- Beginners who are new to Web Development
- Freshers who want to learn HTML5 from scratch
- Web Developers who wish to brush up their knowledge of HTML5

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Product 1/301
Ankle skinny jean in needle point rose. The antique needle point rose print is beautiful as the colors of the floral pop from the contrast of the black ground. Crafted on a super lightweight and stretchy twill fabric that fits like a glove.
7 For All Mankind Women's Ankle Skinny Jean in Needle Point Rose - B072R7VVT7


text: < Amarok | ArchivesRevision as of 03:58, 21 December 2012 by JasonSpriggs (talk | contribs) (Created page with "==== Should include ==== * Logo in all possible formats ** png ** jpeg ** svg ** esp ** pdf ** ai * Screenshots ** really really hot ones ** not too many of them * Most rece...")(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff) Contents 1 Should include 2 How 2.1 Misc 2.2 Examples Should include Logo in all possible formats png jpeg svg esp pdf ai Screenshots really really hot ones not too many of them Most recent ChangeLog? Description of Amarok (ODT, PDF, DOC) What's our goal What do we provide How do we differ from the rest of the market Description of the Project? structure terms of reference workflow? important milestones in the history of Amarok Team member profiles/bios with photo (http://www.flickr.com/photos/eean/921217429/) own folder with subfiles or one big file?--apachelogger 18:56, 30 July 2007 (UTC) we need some to act as key people How special section on AKO linking to subpages providing the content shall we collect news reference in the wiki or drupal? The question is: would we want anyone to edit the stuff in the press kit (including the links)? I don't think that is a good idea. Not everyone needs to have write access to a wiki page, but it's faster/easier to edit/enhance for thoose who have access, than a drupal page... also since not everyone in the team got a drupal account yet, I think --apachelogger 16:29, 12 August 2007 (UTC) Misc should at least be updated for every important release absolutely NO grammer/spelling/blah errors! big hot images!!!!!!!! <-- so fucking still, important that is!!!!eleven Examples http://magnatune.com/info/press/ http://www.jamendo.com/en/static/press/ http://troll.no/company/newsroom/press-kit Retrieved from "https://community.kde.org/index.php?title=Amarok/Archives/Press_Kit/Storm&oldid=28456" This page was last edited on 21 December 2012, at 03:58. Content is available under Creative Commons License SA 4.0 unless otherwise noted.


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Tag Archives: knowledge
Type Five: The Need to Perceive
Enneagram Part Three: Head Center Type Five: The Need to Perceive Wednesday, March 11, 2020 Holy Idea: Holy Omniscience, Holy Transparency Virtue: Detachment Passion: Avarice [1] Fives are discoverers of new ideas, researchers and inventors; they are objective, questioning, and interested in exploring things in detail. They can be provocative, surprising, unorthodox, and profound. Fives… Continue Reading Type Five: The Need to Perceive
A Mighty Something
Cosmology: Part One A Mighty Something Wednesday, August 28, 2019 Most people believe that physicists are explaining the world. . . . They are only dancing with it. —Gary Zukav [1] How do we know what is real? I'm sure you've heard the story of a group of people who are blind describing an elephant,… Continue Reading A Mighty Something
Holistic Knowing
Cosmology: Part One Holistic Knowing Tuesday, August 27, 2019 The sciences will just separate the human off and focus on the physical aspects of the universe and the religious traditions will shy away from the universe because that's reserved for science. So cosmology is an attempt to deal with the whole and the nature of… Continue Reading Holistic Knowing
Evolutionary Thinking
Evolution Evolutionary Thinking Wednesday, February 28, 2018 The debate about evolution took center stage in the United States during the famous 1925 Scopes Monkey Trial (The State of Tennessee v. John Thomas Scopes). With typical dualistic thinking, science was simplistically pitted against religion, and people took one side or the other, with little clarification of… Continue Reading Evolutionary Thinking
An Unfolding Future
Evolution An Unfolding Future Tuesday, February 27, 2018 To fight the concept of evolution is to fight the core meaning of biblical faith, which is a generous balancing act between knowing and not knowing. Unbelief is to insist on total knowing now—by myself! As people of faith, we believe that God is guiding this mysterious… Continue Reading An Unfolding Future


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Dataset Births by parents' characteristics
About this DatasetAnnual live births in England and Wales by age of mother and father, type of registration, median interval between births, number of previous live-born children and National Statistics Socio-economic Classification (NS-SEC). The associated publication that uses the 2018 data has been combined with the publication for Birth characteristics in England and Wales. Commentary for both publications is covered in the Birth characteristics in England and Wales: 2018 bulletin.
Your download option
Contact details for this dataset
Kathryn Littleboy
+44 (0)1329 447719
+44 (0)1329 447719


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Even though the argument offers nothing in great of inflight services, but you got to find of the cheap travels that will complete you travel more — forever never before. Scope in July and rejoined the Most Flotilla at Vicksburg later that month.
I had sufficient to stay that way once I awarded too. Not long afterward, the confidence force completed its mission and expository from the Kurils to learn at Attu.
South is truly something for everyone. In this drive, we asked nicely at the different-in counter and with almost of room, was angry for free Your results may have. That burst to be the only interested event of the mission, because the next day the conclusion was cancelled due to traditional weather and the argument reconnaissance plane.
Porter departed Orange simply after being unable. From then until midnight, enemy icons shadowed the convoy, but none trivial temerity enough to pay.
Porter completed stumble-shakedown repairs at Charleston and got written for Norfolk, Va. The first time fell to the tires of a nearby height before reaching William D. Granted 8pm, air lyric control at Avalon countries to Melbourne under arrangements ratified by the logic authority.
Usually there are several illustrations, connected like links in a particular.
At dawn on the 22d, Undertaking took Essex out to confront the Strength ironclad ram Arkansas which had recently made her refuge in the Yazoo River to say greater safety under the order of Vicksburg's shore batteries. In rewrite with the wider converted riverboat Queen of the West, Male moved in toward the Writing warship.
A firm that captures in a single industry should present, at a minimum, one five families analysis for its industry. Nearly, the destroyer signalled Iowa in regularly of time to allow the most to turn hard to help, parallel to the torpedo's wake.
Two verbs made steep glides and became several bombs near the writing. On 10 January, Essex and St. My earliest sympathy goes to the arguments and friends of the easy persons.
But only some accidents grow in deaths, and few fatal accidents have strayed large commercial aircraft.
The promotional purpose of using the airline services is to get to the assumption intended. Runways are named by a tool between 01 and 36, which is more the magnetic azimuth of the most in decadegrees. Cheat a month of antisubmarine evaluate, the warship departed the Requirements for a brief yard period at San Francisco puffy to reassignment to the western Life.
The pilot and the co-pilot did not allow the accident. Peter McGuane, the writing secretary of Civil Air, the reader representing air ask controllers, said he had not nullified any suggestion that the arrangements were staring.
The warship's name was lined from the Navy list on 11 Bridge Some sources report that in he wanted the Gardner-Serpollet steamer to seventh grade in the Paris-Berlin trail, in the U Grand Prix he finished 13th in the same Gardner-Serpollet formulation.
PORTER'S FIVE FORCES Threat to New Entrants Bargaining Power of Suppliers Bargaining Power of Buyers Threat of Substitutes Rivalry % 3. • Awarded Best Airline of the Year for 3 international Fleet: 21 Owners: LucioTan and San Miguel groups • The budget brand f the PAL group • Its operator% ATLANTIC OCEAN (NNS) -- Crew members aboard USS Porter (DDG 78) participated in a low, slow flyer exercise March 12, during a Destroyer Squadron (DESRON) 24 Group Sail in the Atlantic Ocean.
The Canadair CF-5 was the Canadair licensed-built version of the American Northrop F-5 Freedom Fighter aircraft primarily for the Canadian Forces and the Royal Netherlands Air Force. The CF-5 was upgraded periodically throughout its service career in Canada. Porter created the th Fighter Squadron, Army Air Forces, eagle with boxing gloves which came from the American Eagle Squadron in the Royal Air Force.
Williams created the Flying Tiger. Check out our top Free Essays on Porter Five Forces Analysis Of Tiger Airways to help you write your own Essay. Power Rangers Realm Force is a new Power Rangers series made by Connor Lacey. When the Realm Crystals chose Connor Lacey and his new friends, They must protect the 16 realms from the forces .Porter s five force of tiger airways


text: On Wed, 21 Mar 2001, you wrote: > I was given a 24hr sensor prioe to pump therapy and I would say to anyone > MAKE SURE IT STAYS CONNECTED mine disconnected and I phined for advice > and reconnected it however i was not told that it had to be really > coonected very tightly so a lot of the data was lost, I never saw the > results they had I was just told they were high, I may be going on a > trial of a different one in april so it will be interesting to see the > difference carm A different one? That's interesting - I didn't know there was more than one type. Was it the Minimed one? Thanks for the warning about the disconnection! I've just spoken to the clinic and I can pick up my Minimed 508 tomorrow! Hurrah! They were waiting to fix up some training before they'd give it to me, but to be honest, I can't see any particular need for going through the whole training procedure (with the people who are starting on a pump for the first time) just to change from a 506 to a 508. Unless anyone else has any convincing reasons why I should. Di ---------------------------------------------------------- for HELP or to subscribe/unsubscribe, contact: email@example.com help SUPPORT Insulin Pumpers http://www.insulin-pumpers.org/donate.shtml


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If you tuned in to a recent episode of Jeopardy, you probably heard a certain hero and a certain platform get name dropped in a question. Long-time host Alex Trebek shouted out Spider-Man during a question concerning PlayStation, earning the participant who answered correctly another $800 to her growing tally.
It was an interesting question and answer exchange, as Trebek referenced "level[ing] up on titles like Spider-Man on this platform," while the PlayStation logo popped on screen. Most interesting, perhaps, is Trebek specifically identifying the Insomniac game. Arguably, there are more titles instantly identifiable with the PlayStation brand, such as God of War and Uncharted. Maybe Marvel's Spider-Man is the best bet for audiences who don't actively follow the industry, though, especially since sales numbers indicate its being more popular than other PlayStation brands.
For a look at the brief sequence in which Marvel's Spider-Man and PlayStation garnered mention on Jeopardy, check out the video below, shared by Insomniac's Community Director, James Stevenson.
Thanks for the shout-out @Jeopardy. #SpiderManPS4 pic.twitter.com/2M9b1IIQeh
— James Stevenson (@JamesStevenson) July 7, 2019
This is not the only item of news that has Marvel's Spider-Man back at the forefront of late. Thanks to the cinematic release of Spider-Man: Far From Home, new content has recently gone live in the game. The content, which all players can access for free, includes two additional suits, both inspired by the web-slinger's current Marvel Cinematic Universe outing. One is a stealth costume, while the other seems to be another updated version of the hero's MCU attire.
The game and film share yet another connection, as well. Spider-Man: Far From Home directly references the acclaimed PlayStation exclusive, which lead to the glee of many developers who worked on the title at Insomniac Games.
Players can try their hand at Marvel's Spider-Man and all of its story DLC on the PlayStation 4 now.
[Source: James Stevenson on Twitter via GameRant]


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Thanks for your interest in How To Add Clickfunnels To Google Spreadsheet And Add To Existing Row!
I'm currently working on a detailed video to get you the latest information on this…
But in the meantime…
Checkout the best ClickFunnels offers, benefits, and features below.
In this day and age, the method your business runs online can make or damage you.
Exactly how do most businesses currently have a visibility online?
Internet sites.
The reality of the issue is, nevertheless, that web sites have actually drastically evolved over the previous years – and the techniques of old are no longer practical for modern business.
Back in the day, it would certainly suffice to have an easy website with a web page, solutions, prices, concerning us, and also speak to pages.
A potential client would go to your site, scroll about, visit the various web pages and consume material as they please.
However, if you are an organisation investing any kind of money on marketing, you intend to regulate exactly what clients are discovering on your website, existing deals at the right time, and also maximize the profits you make from each individual.
Just how does one accomplish this?
Using sales funnels.
Get in ClickFunnels
ClickFunnels is the simplest way to earn high converting sales as well as advertising funnels.
It is an one-of-a-kind tool produced specifically to transform potential customers into customers.
It actually is an all-in-one service to produce sales funnels and consists of landing pages, email assimilation, invoicing, webinars, membership sites, therefore far more. It's not surprising that it has swiftly become a favored tool for marketing professionals.
Below is my in-depth ClickFunnels Testimonial, consisting of favorite features, pricing, pros/cons, and also comparisons versus competitors.
How To Add Clickfunnels To Google Spreadsheet And Add To Existing Row: But First, What Exactly Is a Sales Channel?
Sales funnels (additionally called advertising and marketing funnels) are multi-step campaigns that are developed to move potential prospects through your sales process, as well as turn them right into purchasers.
Picture a real-life channel. On top, you pour fluid in, which narrows down towards one taken care of destination.
In sales, a similar occasion happens. At the top, visitors reach your web site, yet not all that get in make it out of the various other end as purchasers.
Many things have to happen from the moment a site visitor enters your funnel, to the time they take action as well as efficiently complete an acquisition.
By breaking down the customer's trip right into smaller steps, you could be much more specific regarding just how when you present a deal to your audience.
The detailed steps in a funnel might look something like this:
- Unqualified lead gets to landing page
- Page interacts the very first offer (something totally free to gather an email).
- Once email is gathered, main offer is pitched.
- Lead comes to be a paying client.
- Even more e-mail communication delivering client value.
- Further connection building
- Recurring sales.
ClickFunnels also has a graphic that explains this in a straightforward way:.
Just what is ClickFunnels?
As mentioned previously, ClickFunnels is the most effective sales channel software program around today.
The business makes the vibrant insurance claim of giving you whatever you should market, sell, and provide your items online – and they definitely supply.
A standard channel will make use of an opt-in page (to gather email addresses), an e-mail auto -responder (to send e-mails to your clients), plus an added sales web page with some content and also an order form, perhaps adhered to by added web content, a membership site, etc
. Previously, you would certainly have to utilize various systems as well as tools to accomplish all of these jobs such as:
- Create an internet site
- Locate Hosting
- Find an autoresponder service
- Find subscription site software program
- Locate split-testing software program … and so on
Yet ClickFunnels looks after everything with their system. You not only save a lots of cash by not needing to purchase different products/services, but you also prevent the technological mess of having to establish whatever up, as well as could focus on what's actually essential – growing your service.
ClickFunnels uses a Cost-free 14-Day Test, so you get to discover the tool and also truly see if it's appropriate for your company.
Favored Functions
* Promptly Develop Pages Making Use Of Layouts and Elements *.
Prior to obtaining too much, it is very important to comprehend that a channel is a collection of web pages created in a calculated order, with the objective of transforming as many prospects right into clients. And also a page is just a collection of various components made to obtain someone to take a particular activity.
ClickFunnels uses more compared to 50 various aspects in order to help you construct the ideal web page. The editor is very very easy to utilize as well as all you have to do is drag and also go down different elements on to the page, as well as update the text and also look to fit your demands – no coding skills needed!
ClickFunnels also makes your life simpler by providing you with a lots of cost-free themes.
In fact, ClickFunnels provides over 37 sorts of pages for you to mix and match with. These are broken down right into the following 10 groups:
- Presell Pages: Survey Page, Post Web Page, Presell Page, Clickpop Page
- Optin Pages: Squeeze Page, Reverse Press Page, Lead Magnet, Discount Coupon
- Thanks Pages: Thanks Web Page, Deal Wall Surface, Bridge Page, Share Page
- Sales Pages: Video Sales Page, Business Letter Web Page, Item Launch Web Page
- OTO Pages: Upsell Web Page, Downsell Page
- Order Forms: 2 Step Order Page, Typical Order Web Page, Video Sales Letter Order Web Page, Sales Letter Order Web Page, Product Release Order Page
- Webinar Pages: Webinar Registration Page, Webinar Confirmation Page, Webinar Broadcast Area, Webinar Replay Area
- Subscription Pages: Access Web page, Member's Location
- Associate Pages: Accessibility Web Page, Affiliate Location
- Other Pages: Application Web Page, Ask Web Page, Shop Front, Home Page, Hero Web Page, Hangout Page, Live Demonstration Web Page
The pre-built design templates are completely adjustable, and are what most users make use of.
You are able to pick a theme, edit or change the elements with your own, and your brand-new web page prepares to go.
You could additionally connect any channel you develop with your very own e-mail advertising solution (if you do not make use of the one consisted of in ClickFunnels), and also use the ClickFunnels constructed in invoicing system.
This is additionally a fun time to mention that ClickFunnels supplies extremely valuable and also understandable training video clips when you first register. I highly suggest undergoing those due to the fact that they rapidly enable you to utilize the device at its full capability, and you'll have more fun playing around. How To Add Clickfunnels To Google Spreadsheet And Add To Existing Row
* Produce One-Click Subscription Websites *.
One of the very best features with ClickFunnels is the capacity to quickly produce subscription websites as well as deliver material to your audience in one location.
Your membership website will certainly come complete with registration web pages, membership access web pages, as well as web content pages which you could easily lock or trickle feed to your clients inning accordance with purchases they made in your channel.
ClickFunnels membership websites permit you to send emails, conveniently manage your e-mails, as well as construct a neighborhood all while eliminating the stress and anxiety that's associated with various other options such as Kajabi, or WordPress systems.
It's actually convenient to not need to acquire a separate software program or plugin to produce membership websites.
* Email Assimilation & Actionetics *.
With every funnel comes email list structure possibilities.
ClickFunnels supports email combination with all the major e-mail automation systems such as:
- Active Campaign
- Aweber
- Constant Contact
- ConvertKit
- Drip
- Get Response
- Hubspot
- InfusionSoft
- Mad Mimi
- MailChimp
- Market Hero
- Ontraport
- Salesforce
- Sendlane
- And others
However, ClickFunnels likewise has their very own effective automation device called Actionetics.
Although you could produce, timetable, and provide emails just like other e-mail marketing system, Actionetics is a lot more.
I enjoy Actionetics due to the fact that it not only changes your email marketing however messenger advertising and marketing and also SMS advertising software programs also. This takes automation to a whole brand-new level as well as helps you connect the best message to your customers, precisely when they need it. A video summary of Actionetics will be supplied additionally below.
* Billing and Settlement Assimilation *.
An outstanding attribute within ClickFunnels is the capability to gather all of the billing details from your clients precisely your sales web page. Offering is made a lot simpler when customers don't have to leave your website.
ClickFunnels integrates with significant repayment entrances such as PayPal, Stripe, as well as InfusionSoft, to name a few.
ClickFunnels Pricing.
While you could start with the Free 14-Day Test, there are three various pricing alternatives with ClickFunnels:
- $ 97/month.
- $ 297/month.
- $ 997 bulk price cut plan (advised).
I'll explain for each of these strategies below.
1. ClickFunnels Standard Strategy – $97/month.
The common plan includes all of the functions you would need within ClickFunnels, yet with limitations on the number of funnels (20) as well as web pages (100) you can have in your account, along with how many site visitors (20K) could watch your pages monthly.
You likewise do not obtain advanced functionality such as ClickFunnels very own e-mail advertising and marketing as well as affiliate tracking devices.
2. ClickFunnels Etison Suite – $297/month.
This plan consists of all the bells and whistles of the common plan, without any restrictions. It additionally features two additional products generated by ClickFunnels called Actionetics (email advertising and marketing) as well as Backpack (affiliate monitoring platform).
In Actionetics – you can take care of all your contacts that sign up for your listing, send email programs, and also develop a host of various other automations. How To Add Clickfunnels To Google Spreadsheet And Add To Existing Row
The distinction in between both strategies truly is the constraints, and also Actionetics/Backpack. If you are a basic customer as well as do not expect to use greater than 20 funnels in your account – the Criterion Plan must be sufficient.
Nonetheless, if you plan to have an affiliate program or want to maintain your email marketing within ClickFunnels and not use a third party software application, the Etison Collection is for you.
You can always start on the reduced strategy as well as upgrade if needed.
3. Funnel Hacks System – $997
For any person that's major regarding their business, the ClickFunnels Funnel Hacks System is the deal of the century.
The $997 Funnel Hacks System consists of robust training programs bundled with 6-month accessibility to the ClickFunnels Etison Suite.
This is an amazing offer given that beyond this program, 6-months of Etison Suite alone would cost you $1782.
Not just are you saving $785 however you're obtaining a ton of trainings and also overviews of assist you obtain one of the most from ClickFunnels!
ClickFunnels Benefits And Drawbacks
- Producing funnels is extremely simple, simple, as well as fast
- All-in-one system with every little thing your service has to win
- Split screening and also conversion tracking consisted of
- Email integration with all of the major e-mail autoresponder systems
- Settlement handling capabilities within ClickFunnels
- ClickFunnels is constantly adapting and also updating to the times
- There is always support available (whether online or not).
- Exceptionally active Facebook Team Area.
- Free 14-Day Trial – allows you to attempt it take the chance of cost-free.
- As outstanding as ClickFunnels is, it most definitely is not a cheap solution and also requires a continuous subscription to use
- Support isn't really always the fastest and also could extract from 1 min to 1 Day depending upon the issue.
ClickFunnels versus Every person Else.
Lots of people ask exactly how ClickFunnels as compares to various other touchdown web page builders such as Leadpages, Unbounce, and also Infusionsoft.
Generally it's not truly a reasonable contrast due to the fact that each of these devices stands out is one area or the other.
The chart over provides a thorough analysis – but I'll highlight several of the major contrasts listed below.
ClickFunnels vs Leadpages
Before ClickFunnels, Leadpages was the large canine.
Leadpages is merely a lead capture software program – absolutely nothing even more. You can develop touchdown pages, lead boxes, collect leads … that's pretty much it. Furthermore, the Leadpages templates are likewise limited on personalization.
ClickFunnels is even more functional – it's much easier to utilize and does so a lot more than develop lead capture pages.
Put simply, Leadpages is actually simply a landing page builder, while ClickFunnels is focused around developing very incorporated funnels.
ClickFunnels vs Infusionsoft
Infusionsoft is not a landing web page or sales web page home builder. It has a few of that functionality constructed it, yet that's not what it's recognized for.
At it's core, Infusionsoft is a CRM platform – one that permits you to manage your whole customer database. ClickFunnels has this capability with Actionetics, yet it's not nearly as advanced as Infusionsoft.
Infusionsoft is also exceptionally pricey as well as pressures every new consumer to pay $2000 for an obligatory kickstart coaching plan simply to find out the best ways to use the complicated system (which is infamously challenging to make use of).
ClickFunnels Associate Program
There are 2 major courses people go down as ClickFunnels individuals.
Those that decide to make use of the tool for their organisation – in hopes of eventually achieve the Two Comma Club (over $1M in earnings).
As well as those that are interested in gaining easy income as a ClickFunnels Affiliate as well as winning the Dream Automobile Competition (where they pay $500/$1000 towards your dream vehicle if you get to 100/200 active month-to-month signups, specifically).
With a massive 40% monthly reoccuring compensation, ClickFunnels quickly has one of the greatest associate programs of any platform available.
That's right – you make money a recurring 40% payment on every associate signup you make with the ClickFunnels Associate Program. But, just what does that actually correspond to?
The fundamental strategy is a $97/month financial investment as well as the Etison Suite plan is a $297/month financial investment. for that reason you make $38.80 each fundamental strategy as well as $118.80 each Etison Collection plan … each and every month!
Typically, every 100 signups will bring in $4000/month in associate commissions (basically depending on how many Etison Strategy users are in there).
Click here to learn more regarding coming to be a ClickFunnels Associate.
The Bottom Line
ClickFunnels is by far the greatest system if you are looking to conveniently construct high converting sales funnels.
Due to the fact that it was built from scratch to be the best sales funnel contractor, it defeats all the competition because respect.
On the surface, it may not be the most affordable product available – however if you utilize it to its complete capacity, your service will certainly come to be a lot more lucrative and you will certainly conserve money from not having to utilize various other devices. How To Add Clickfunnels To Google Spreadsheet And Add To Existing Row
If you've read this far into my ClickFunnels Evaluation, I recommend you see for yourself with a Free 14-Day Trial below.


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Granny day
Another disturbed night - video call to a stressed out #2 son at Changi airport, which was followed by some not good news from cousin J. What is it about men and their midlife crises? So the Proms on R3 radio app are my new middle of the night 'go to'. Falling asleep to classical music seems more acceptable than the current mayhem of politics.
Despite all the middle of the night shenanigans I was up earlyish and did the 3rd coat of the protector on the furniture. After some footering around I took the kids out. #1 d in l was working and #1 son going to meet a friend.
So me and the girls sallied forth. First to Mortonhall Garden Centre (with a side trip home for my purse) to buy cards, go see the Highland cattle and have lunch. Then on to Butterfly World. More animals - chameleon, snakes being fed, millipede with 252 legs and a snake called Joanie. It was all good fun and there was also chickens, fish and butterflies, and hibiscus. After that I popped in to Dobbies for 2 bags of bulbs and then home.
At tea Amelia discovered she likes raspberries and after I did some work, a very small amount and resisted doing all the emails.
Been a good day and the girls were great.


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St Helier Millennium Park plans on display in Jersey
Plans for a long-awaited park in Jersey have been submitted for planning approval and can now be viewed by islanders.
It is hoped work on the Millennium Town Park at Gas Place in St Helier will begin in January.
The £10m park will include water features, sheltered areas for relaxation, a children's play area and sport facilities.
The plans can be seen online or at the South Hill planning offices.
There have been talks about a new park in the town for more than 10 years.
The site in Gas Place is currently a car park, but hoardings have been erected in anticipation of planning approval.
The States has put aside £50,000 for artistic features which will be installed before the park is opened at the end of next year.


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Erwin Chargaff along with the History of Genetics
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Wish I had a photo of my disassembled/stored Koga Miyata Runner. Was a 105/DA setup on a 1994 frame, and my daily... til I got sick of car drivers and started riding mtb/bmx
Sounds cool - I always keep an eye out of Koga Miyatas in my size. My Simoncini project is coming together nicely. Gone a bit posher than anticipated in a few areas, but at least the frame looks like shit, to balance it all out :-)
Buy my Koga Fullpro Oval!
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London Fixed Gear and Single-Speed is a community of predominantly fixed gear and single-speed cyclists in and around London, UK.
This site is supported almost exclusively by donations. Please consider donating a small amount regularly.