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Introns excised from the Delta primary transcript are localized near sites of Delta transcription

Introns excised from the primary transcript of Delta (Dl), a Drosophila neurogenic gene, accumulate to unusually high levels in embryos. High resolution in situ hybridization reveals a striking localization of the excised introns to two foci per embryonic nucleus. The number of foci can be altered by varying the number of Dl genes present in the embryonic nucleus, suggesting that the excised introns are localized near sites of Dl transcription. This conclusion is supported by the observation that larval and imaginal disc nuclei containing two copies of Dl exhibit only one focus of intron accumulation, as expected for nuclei in which homologous chromosomes are paired. Interestingly, the excised introns do not appear to diffuse away from the foci until late prophase, at which time the foci disperse into numerous small dots of hybridization. These results suggest that the excised Dl introns may be associated with a structural element within the nucleus that is dissociated during cell division.

M ost genes in higher eukaryotes contain introns that are transcribed into pre-mRNA and subsequently removed by RNA splicing. The ability to reproduce RNA splicing in vitro has permitted a detailed characterization of many of the steps involved in this process (reviewed in Maniatis and Reed, 1987;Sharp, 1987;Guthrie and Patterson, 1988;Ruby and Abelson, 1991). However, little is known about the mechanics of RNA processing as it occurs within the highly structured nucleus. For example, the mechanism by which pre-mRNAs and excised introns are retained in the nucleus while mRNAs are exported is currently unknown. Similarly, the process of snRNP "recycling; which requires the dissociation of snRNPs from excised introns, is poorly understood.
A number of laboratories have characterized the nuclear distribution of components of the splicing machinery to gain insight into the relationship between RNA processing and nuclear structure. Immunofluorescent staining with anti-snRNP antibodies has revealed 20-50 discrete regions within the nucleus where snRNPs are concentrated, as well as low levels of snRNPs in the surrounding nucleoplasm (Lerner et al., 1981;Spector et al., 1983;Reuter et al., 1984;Nyman et al., 1986). Recently, a non-snRNP factor required for spliceosome assembly was shown to be localized specifically to the regions of snRNP concentration, suggesting that these regions may represent active splicing compartments within the nucleus (Fu and Maniatis, 1990). Immunoelectron microscopic analyses further suggest that these putative splicing compartments form a reticular net-Casey C. Kopczynski's current address is Department of Molecular and Cell Biology, University of California, Berkeley, California 94720. work that extends from the center of the nucleus to the nuclear periphery (Spector et al., 1983;Fakan et al., 1984;Spector, 1990).
Additional evidence for a functional relationship between nuclear structure and RNA processing has come from the localization of specific pre-mRNAs within the nucleus. The nuclei of cells latently infected with Epstein-Barr virus (EBV) accumulate unprocessed EBV RNA along "tracks" that extend from the site of transcription to the nuclear periphery (Lawrence et al., 1989). It has been suggested that these "tracks" may represent specific channels for RNA splicing or mRNA export within the nucleus. In addition, a potential role for the nuclear lamina in RNA processing has been suggested based on the observation that acetylcholine receptor (AChR) intronic sequences are localized at the nuclear periphery in myoblasts (Berman et al., 1990).
Further insight into the spatial organization of the splicing machinery could be obtained by determining where introns accumulate after they have been excised from pre-mRNA. For example, excised introns might accumulate along "tracks" similar to those observed for EBV RNA if splicing occurs during transport to the nuclear periphery. Alternatively, the distribution of excised introns might reveal specific nuclear sites where snRNP "recycling" occurs. To address this issue, we have taken advantage of an unusual feature of the Drosophila Delta (D1) gene: introns excised from the DI primary transcript accumulate to levels readily detectable by RNA blot analysis and in situ hybridization (Kopczynski et al., 1988;Kopczynski and Muskavitch, 1989;Haenlin et al., 1990;this report). We find that the accumulation of excised DI introns is highly localized within the nucleus near sites of Dl transcription, indicating that splicing of the DI pre-mRNA occurs near the DI gene. Fur-thermore, we observe that this localization of excised introns is disrupted during mitosis at late prophase, just before the reported dispersal of many nuclear matrix-associated antigens (reviewed in Verheijen et al., 1988). The implications of these results for models of RNA processing within the nucleus are discussed.

RNA Blot Hybridization
Preparation of total embryonic RNA and oligo(dT)-selection of poly(A) + RNA were performed as previously described (Kopczynski et al., 1988), except total RNA preparations were further purified by LiC1 precipitation (Sambrook et al., 1989). RNA samples were resolved on formaldehyde/agarose gels and subjected to partial alkaline hydrolysis (Maniatis et al., 1982) before transfer to BioTrans nylon membranes (ICN, Costa Mesa, CA). Blot hybridization and 32p labeling of DNA probes were performed as described by Feinberg and Vogelstein (1983). The lengths of the RNA species identified were estimated from their mobility relative to a 0.24-9.5kb RNA ladder (BRL, Gaithersburg, MD).

In Situ Hybridization
RNA probes were internally labeled with digoxygenin-UTP (Boehringer Mannheim Biochemicals) by in vitro transcription according to the manufacturer's instructions. Probes were reduced in length to ,'o100-300 nucleotides by incubating for 10 rain in 1 M sodium carbonate, pH 10.2, at 60~ Hybridization of the probes to whole, paraformaldehyde-fixed embryos was performed as described in Tautz and Pfeifle (1989), except 5% (wt/vol) dextran sulfate (Pharmacia Fine Chemicals, Piscataway, NJ) was included in the hybridization solution, and the hybridization was performed at 52~ for 12-18 h. Embryos were subsequently washed at 52~ for 6-18 h in five changes of hybridization solution (without dextran sulfate), then for 30 rain in PBT (130 mM NaC1, 10 mM sodium phosphate, pH 7.2, 0.1% [vol/vol] Tween 20). To visualize the hybridized probe, embryos were incubated at room temperature for 1.5 h with HRP-conjugated antidigoxygenin (150 U/ml; Boehringer Mannheim Biochemicals) diluted 1:200 in PBT + 5 % (vol/vol) normal goat serum, washed for 1.5 h in four changes of PBT, then incubated with 0.5 mg/ml 3,3-DAB, 0.003% H202 in PBT until the embryos were light brown in color. After three brief washes in PBT, the DAB precipitate was rendered opaque by silver precipitation (Gallyas et all., 1982;Liposits et al., 1984). Embryos were mounted in methyl salicylate for microscopy or stained with 1 #g/ml Hoechst 33342 for 10 min and mounted in 80% (vol/vol) glycerol in H20.
The same protocol was used for in situ hybridization of digoxygeninlabeled probes to larval tissues and imaginal discs, except that heptane, required during embryo fixation to permeabilize the vitelline membrane, was usually omitted from the fixation step.
We have furthered our characterization of the minor RNAs by mapping the genomic positions of DNA fragments that hybridize specifically to the minor RNAs. Fig. 1 A compares the minor RNA "hybridization map" to the intron/exon map of the Dl mRNAs (Haenlin et al., 1990;Fechtel, K., K. A. Bauer, and M. A. T. Muskavitch, manuscript in preparation). The results show that all of the genomic fragments that hybridize to the minor RNAs are located within Dl intronic regions. Most importantly, the length of each of the five minor RNAs estimated from RNA blots is very close to the length of the Dl intron with which it hybridizes (Figure 1 B). This suggested that the minor RNAs may, in fact, be excised introns. To test this hypothesis, the 5' ends of the 3.7-and 2.2kb minor RNAs were mapped by primer extension analysis ( Fig. 1 C). The 5' end of the 3.7-kb RNA maps precisely to the exon 3/intron C donor splice site, while the 5' end of the 2.2-kb RNA maps one nucleotide downstream of the exon 5/intron E donor splice site. We also found that the 5' ends of the 3.5-and 2.8-kb minor RNAs map to the exon 2/intron B donor splice site and the exon 4/intron D donor splice site, respectively (data not shown). These data strongly support the conclusion that the minor RNAs are introns excised from the Dl primary transcript. The alternative possibility, that the minor RNAs are transcribed from independent promoters located at the splice junctions, is highly unlikely since transcription of the minor RNAs and the Dl mRNAs requires the same upstream promoter region (Haenlin et al., 1990). The conclusion that the minor RNAs are excised introns is further supported by the restricted accumulation of the minor RNAs within the nucleus (see below). We estimate the steady state level of each excised intron to be ,~5 % of Dl mRNA levels based upon RNA blot analysis performed using total (unfractionated) embryonic RNA ( Fig. 1 B, lanes DI1 and A3, and data not shown). Since excised introns are usually rapidly degraded in the nucleus, the accumulation of excised Dl introns suggests that these introns have relatively long half-lives.

Excised DI Introns Are Highly Localized within the Nucleus
We could not determine from our previous in situ hybridization data whether the excised Dl introns might be localized to a specific region of the nucleus, as has been reported for  (Kopczynski et al., 1988); (A3, B4, C4, D3) representative genomic fragments derived from introns A, B, C, and D, respectively; (E/) genomic fragment containing sequences from the 2.9-kb intron, 2.2-kb intron, exon 5, and exon 6. The approximate length of each RNA species detected is indicated. The high molecular weight transcript in lane E1 (arrowhead) is only observed when probes containing exon sequences are hybridized to oligo(dT)-selected RNA; it is probably an incompletely spliced pre-mRNA. (C) Primer extension analysis. Primers 3.9-1 and 2.2-1 were end-labeled with 32p, hybridized to 50 #g oligo(dT)-selected RNA, then extended with reverse transcriptase. The extension products were resolved on gels next to sequencing reactions primed by the same oligonueleotide. The arrowheads indicate the positions of the 3.9-1 and 2.2-1 primer extension products relative to the exon 31intron C and exon 5/intron E splice site sequences, respectively. (Uppercase letters) Exon sequence; (lowercase letters) intron sequence. Sequencing reactions were loaded in the order "GATC" from left to right.

Figure 2.
Excised introns accumulate at highly localized foci within embryonic nuclei. Whole embryos were hybridized with digoxygeninlabeled antisense RNA. After washing, the hybridized probe was detected histochemically using a HRP-coupled anti-digoxygenin antibody. The histochemical reaction product was rendered opaque by silver precipitation to increase the sensitivity of the technique. (A and B) Gastrulation stage embryos hybridized with a D11 cDNA probe (A) or an intron-specific probe generated from genomic fragments A2-D3 ( Fig. 1 A) (B). This intron probe has a potential sequence complexity of 12 kb and thus provides an optimal hybridization signal. (C) Optical cross section through nuclei of a precellularization stage embryo hybridized with the intron probe. (D) Optical sagittal section through nuclei of a cellularization stage embryo hybridized with the intron probe. (n) Nucleus; (c) cytoplasm; (y) yolk. (E) Radial distribution of 124 intron accumulation sites from 73 precellularization stage nuclei superimposed on a single nucleus. Each site was placed on the diagram based upon its distance from the nuclear envelope relative to the diameter of the nucleus in which it was located. Measurements were only recorded when the accumulation site and the nuclear membrane were in the same focal plane. The dotted lines delimit a region within which the density of accumulation sites is threefold greater than within the surrounding area. Bars: (A and B) 50 #m; (C) 5/~m; (D) 10 #m.
EBV and AChR RNAs. We therefore repeated our analysis using a nonradioactive in situ hybridization protocol (Tautz and Pfeifle, 1989) to achieve greater resolution than previously possible with 35S-labeled probes. We modified the in situ hybridization protocol described by Tautz and Pfeifle (1989) to include a silver precipitation step (Gallyas et al., 1982;Liposits et al., 1984), which renders the normally translucent histochemical reaction product opaque. This modification greatly enhances the sensitivity of the technique. Fig. 2 shows the different hybridization patterns observed in wild type embryos using a Dl cDNA probe and a probe specific for introns A-D. In contrast to the Dl mRNA, which accumulates predominantly in the cytoplasm (Fig. 2 A), the introns accumulate specifically at two foci within each embryonic nucleus (Fig. 2, B-D). Two foci are also observed using a probe that recognizes intron C alone (data not shown), suggesting that all Dlintrons accumulate in the same two regions within each nucleus. A fraction of the signal observed probably corresponds to incompletely spliced pre-mRNA, since low levels of high molecular weight RNA are detected when intron probes are hybridized to total embryonic RNA (Fig. 1 B, lane A3). However, the majority of the hybridization signal observed on total RNA blots probed with intron probes corresponds to excised introns ( Fig. 1 B and data not shown), strongly suggesting that the majority of the signal observed in the nucleus represents hybridization to excised introns. Control hybridizations using sense-strand probes of similar complexity or RNAse-treated embryos revealed no detectable hybridization of the intron probe to Dl chromosomal DNA (data not shown).
The excised intron foci often appear to be located near the nuclear envelope (Fig. 2, C and D). Since it has been suggested that RNA splicing may occur in association with nuclear pores (Btobel, 1985;Urlaub et al., 1989;Berman et al., 1990), we were interested in determining if this localization occurs more frequently than expected for a random distribution of foci. To address this question, the distances between the nuclear membrane and each of 124 foci (from 73 embryonic nuclei) were measured relative to the diameter of the nucleus. These values were then used to superimpose the foci onto a single model nucleus. Fig. 2 E shows that the distribution of foci within the embryonic nucleus is not random; the density of foci is threefold higher in the area between the dotted lines than in the surrounding areas. However, it is unlikely that this localization results from an association between RNA splicing and nuclear pores since this region is not adjacent to the nuclear membrane. We also note that 30% of the foci are located outside of this region, indicating that the sites of intron accumulation are not restricted to a specific domain within the nucleus.

Excised Dl lntrons Accumulate Near Sites of Transcription
Since excised introns remain associated with spliceosomal snRNPs (Pikielny et al., 1986;Cheng and Abelson, 1987;Konarska and Sharp, 1987), the excised intron foci could represent specific sites of spliceosome disassembly and intron turnover within the nucleus. Alternatively, the excised DI introns might be accumulating at sites of Dl transcription, as suggested by the presence of two foci within each nucleus. To distinguish between these possibilities, we tested whether the number of foci observed within the nucleus is dependent upon the number of DI genes present. Fig. 3 shows that embryos heterozygous for a deficiency of Dl have only one site of intron accumulation per nucleus (Fig. 3 A), whereas embryos heterozygous (Fig. 3 C) or homozygous (Fig. 3 D) for a duplication of Dl (Dp(3;3)bxd~~ Lindsley and Grell, 1968) have three and four sites of intron accumulation per nucleus, respectively. Thus, the number of foci corresponds precisely to the number of DI genes present in the nucleus, suggesting that the introns accumulate at sites of DI gene transcription.
As an additional test of this hypothesis, the intron probe was hybridized to embryos carrying a DI transposon on the second chromosome. Genetic analyses indicate that the DI gene carried by the transposon is expressed at lower levels than the wild type Dl gene (Kopczynski, 1991). Fig. 3 (E and F) show that embryos carrying one copy of the wild type Dl gene and two copies of the DI transposon have one large focus and two smaller loci of hybridization per nucleus. The number and sizes of the foci suggest that they represent the wild type gene and the two DI transposons, respectively.
These data strongly support the conclusion that the excised DI introns accumulate near sites ofDl gene transcription. We also note that the Dl transposons (Fig. 3 F, arrows) appear to be located at a more basal position within the nucleus than the wild type DI gene (Fig. 3 E, arrows). This probably reflects the chromosomal positions of the transposon and the wild type DI gene relative to their respective centromeres, since chromosomes in these nuclei are oriented with centromeres positioned apically and telomeres positioned basally (Foe and Alberts, 1985).

Physical Evidence for Pairing of Homologous Chromosomes in Imaginal Nuclei
Homologous chromosomes are physically paired in larval polytene nuclei, and genetic experiments have suggested that they are also paired in imaginal nuclei (Lewis, 1954;Garcia-Bellido and Wandosell, 1978;Jack and Judd, 1979;Gelbart, 1982). If the excised introns are localized at sites of Dl transcription, the intron foci should be colocalized in larval and imaginal nuclei since both copies of the Dl gene should be immediately apposed within the nucleus. Fig. 4 A shows that wild type eye imaginal disc nuclei that express Dl contain only one site of intron accumulation, even though two Dl genes are present. Similarly, eye imaginal disc nuclei homozygous for a Dl duplication (four copies of D/) contain only two sites of intron accumulation (Fig. 4 B), as do polytene nuclei of the larval trachea that contain four copies of DI ( Fig. 4 C). We have observed that the two sites of intron accumulation in the large polytene nuclei are always relatively close to one another, consistent with the fact that the DI duplication is located on the third chromosome only eight map units away from the wild type DI gene. These data further support the conclusion that the excised DI introns accumulate specifically at sites of DI transcription and repre-

Mitosis Disrupts Excised Intron Localization
A transient lack of intron hybridization is observed in specific embryonic domains during early germ band elongation (Fig. 5 A). The positions of these domains and the timing with which they appear correspond well with the domains of mitotically active cells present during this stage of embryogenesis (Foe, 1989). At higher magnification, it is evident that some nuclei within these domains contain small "dots" of hybridization in addition to the two relatively large foci normally seen during interphase (Fig. 5 B). We also observe nuclei in these domains that contain two foci of reduced size and 20-30 "dots" of hybridization, nuclei with two or four small foci and no detectable "dots" and cells without any detectable hybridization (see below).
To determine whether the changes in Dl intron distribution are correlated with different stages of mitosis, we used a DNA-specific dye to visualize chromosomes in embryos hybridized with the intron probe, Fig. 5 reveals that during early prophase (Fig. 5, C and D, large arrow), introns remain highly localized within the nucleus. By late prophase (Fig. 5,C and D,small arrows), however, small "dots" of hybridization become apparent in addition to the larger foci. The larger foci appear to be localized with the condensing chromosomes at this stage (Fig. 5, C and D), while the smaller "dots" are often spatially separate from the fluorescent DNA (data not shown). During metaphase, the small "dots" are no longer detectable (Fig. 5, E and F, large arrow).
Instead, two pairs of small loci are visible over the condensed chromosomes. The four small foci present during metaphase are further reduced in size by late anaphase and appear to have segregated with the chromosomes such that two foci are now present at each spindle pole (Fig. 5, E and F, small arrow). These foci may represent nascent pre-mRNA trapped by chromosome condensation, or excised introns that become peripherally associated with the condensed chromosomes. By telophase, hybridization of the intron probe is no longer detectable (Fig. 5, G and H). These data indicate that the dispersal and apparent degradation of the excised introns is a highly ordered process which, based upon the time required to complete mitosis at this stage (Foe, 1989), is completed in <5 min at 25~

Discussion Nuclear Accumulation of Excised Dl Introns
We have shown that the five minor Dl RNAs present in oligo(dT)-selected RNA preparations are excised Dl introns.
This conclusion is based upon the correlation between the lengths of the introns and the lengths of each of the minor RNAs, the fact that the 5' ends of the 3.7-and 2.2-kb minor RNAs map to the predicted intron splice junctions, and the accumulation of the minor RNAs within the nucleus. The results of Haenlin et al. (1990), which also revealed that the transcription of all Dl RNAs requires the same promoter region, led these authors to similarly conclude that the minor RNAs are excised introns. Since excised introns are not likely to be substrates for polyadenylation, their retention on oligo(dT) columns is probably due to internal adenylate-rich sequences. We could not determine whether the excised introns are in a lariat or linear conformation because their large sizes prevented the use of PAGE to characterize their structure (Grawbowski et al., 1984;Ruskin et al., 1984). However, a number of investigations have shown that excised introns exist predominantly as lariats in vivo (Domdey et al., 1984;Rodriguez et al., 1984;Zeitlin and Efstratiadis, 1984;Sittler et al., 1987;Zeitlin et al., 1987). In fact, excised intron lariats are protected from degradation in yeast mutants that lack debranching activity (Chapman and Boeke, 1991), indicating that the usual rapid degradation of excised introns occurs after debranching. Given these results, it is likely that the excised DI introns accumulate predominantly as lariats.
The relatively high levels of excised intron accumulation could reflect a specific property of the Dl introns or a more general property of the splicing machinery in Drosophila cells.
The excised D! introns accumulate at highly localized loci within the nucleus. We have not detected any accumulation The Journal of Cell Biology, Volume 119, 1992 of the excised introns outside of these foci during interphase, even in nuclei that contain four copies of the Dl gene. However, by using silver precipitation to enhance the visibility of the histochemical reaction product, we were able to detect dispersal of excised DI introns during mitosis. This observation underscores the sensitivity of the in situ hybridization technique used, and it suggests that the excised introns are in some way immobilized during interphase. Since excised /5-globin introns have been shown to be tightly associated with isolated nuclear matrices (Zeitlin et al., 1987), it is possible that the excised DI introns are bound to an equivalent structure in vivo. The fact that the excised introns become dispersed at late prophase, just prior to the dispersal of many nuclear matrix-associated antigens (reviewed in Verheijen et al., 1988), supports this possibility. Our observation that the number of Dl genes present within the nucleus determines the number of excised intron foci observed strongly suggests that the excised introns accumulate near the sites of D! transcription within the nucleus. Without exception, embryonic nuclei that contain a single DI gene exhibit a single focus of excised intron accumulation, while embryonic nuclei containing two, three, or four DI genes exhibit two, three, or four foci, respectively. In imaginal discs, however, where homologous chromo-somes are believed to be paired, nuclei that contain two Dl genes exhibit only one focus of excised intron accumulation, while nuclei that contain four Dl genes exhibit only two excised intron foci. The conclusion that the excised introns accumulate near sites of DI transcription is further supported by the observation that the foci colocalize with condensing chromosomes during mitosis.
The accumulation of excised introns near sites of DI transcription is consistent with previous electron microscopic and biochemical analyses of in vivo splicing in Drosophila. Beyer and Osheim (1988) used EM to characterize spliceosome assembly and intron removal from nascent transcripts in embryonic nuclei. These authors found that introns located >4 kb upstream of a polyadenylation site are frequently removed cotranscriptionally. Similar results have been reported by LeMaire and Thummel (1990), who used biochemical techniques to analyze splicing at the E74A gene of Drosophila. It therefore seems likely that the accumulation of excised introns near sites of DI transcription is a consequence of splicing being completed before the Dl pre-mRNA is released from the DNA template.
Recently, Shermoen and O'Farrell (1991) have reported the use of intron probes to follow transcription of the Ultrabithorax (Ubx) gene through the cell cycle in Drosophila embryos.
These authors observed two foci of intron accumulation in interphase nuclei using a probe for the 5'-most Ubx intron and noted that these foci disappear during mitosis. Based on the assumption that an excised intron would be rapidly degraded, the authors propose that the disappearance of the foci reflects abortion of the nascent Ubx transcripts. Our results, along with the demonstrations of cotranscriptional splicing by Beyer and Osheim (1988) and LeMaire and Thummel (1990), suggest that the hybridization signal observed by Shermoen and O'Farrell (1991) could predominantly represent excised 5"most Ubx introns, not nascent Ubx transcripts. If so, then much of the decrease in hybridization observed during mitosis might be the result of the dispersal of excised Ubx introns at prophase, as we have observed for the excised DI introns.

Interphase Pairing of Homologous Chromosomes in Imaginal Nuclei
The ability to visualize the location of DI genes within the nucleus allowed us to address whether homologous chromosomes are paired during interphase in diploid Drosophila cells. Our results reveal that Dl genes located on homologous chromosomes are colocalized in imaginal disc nuclei. This provides physical evidence for homologous chromosome pairing during interphase in imaginal cells, which previously has been inferred from genetic analyses (Lewis, 1954;Garcia-Bellido and Wandosell, 1978;Jack and Judd, 1979;Gelbart, 1982). The mechanism responsible for the pairing is still unknown, but its effect on gene regulation in Drosophi/a has been well-documented (Lewis, 1954;Kaufman et al., 1973;Jack and Judd, 1979;Babu and Bhat, 1981;Gelbart, 1982;Gelbart and Wu, 1982;Gubb et al., 1986). Interestingly, pairing-dependent genetic complementation, or'transvection" (Lewis, 1954), has not been observed in Drosophila embryos. This is consistent with our discovery that homologous Dl genes are physically separate in embryonic nuclei, suggesting that homologous chromosomes are not paired during early development.

Models for the Relationship between Nuclear RNA Processing and Transport
The "gene gating" (Blobel, 1985) and "translational translocation" (Urlaub et al., 1989) models for gene expression and RNA processing propose that the splicing machinery is associated with nuclear pores. The distribution ofDl intron accumulation sites within the nucleus demonstrates that Dl transcription and pre-mRNA processing do not require association with the nuclear lamina, and thus does not support these models. However, Spector (1990) has recently proposed a model for the organization of the nucleus that is consistent with our results. The model, based upon immunohistochemistry and 3H-uridine labeling of nuclei, proposes that the splicing machinery is organized as a reticular network that extends throughout the nucleus (excluding nucleoli) and that actively transcribed genes are peripherally associated with this network. According to this model, the different sites of Dl intron accumulation within the nucleus represent different positions of the Dl gene along this reticular network.
The localization of excised Dl introns near sites of Dl transcription differs from the localization of cardiac ot-actin and AChR introns in chick myoblast nuclei. Berman et al. (1990) have shown that the fifth intron of the a-actin pre-mRNA is widely distributed in myoblast nuclei, whereas the first intron of the AChR pre-mRNA is specifically localized around the nuclear periphery. (We note that the authors did not determine whether these distributions represented excised introns or unspliced pre-mRNA). We suggest that the different intranuclear distributions of Dl, o~-actin, and AChR intronic sequences could reflect different rates of pre-mRNA splicing relative to nuclear transport. Since all of the DI introns are more than 4 kb upstream of the predominant polyadenylation site (Kopczynski et al., 1988;Haenlin et al., 1990), the accumulation of excised DI introns near sites of DI transcription is probably a consequence of splicing being completed on the nascent pre-mRNA (discussed above). In contrast, the 3' end of the fifth o~-actin intron is less than 800 bp from the polyadenylation site (Chang et al., 1985). If the rates of pre-mRNA splicing in chick myoblasts and Drosophila embryos are similar, then the fifth c~-actin intron would not be removed until after the pre-mRNA is released from the DNA template. The final splicing of the ct-actin pre-mRNA might then occur as the immature transcript is transported to the nuclear envelope, resulting in a wider distribution of cr intronic sequences. The perinuclear distribution of the first AChR intron suggests that this intron is not removed from the pre-mRNA until after transport to the nuclear envelope is completed. This could be the result of inefficient recognition of this intron by the splicing machinery, perhaps due to unusual secondary structure or nonconsensus splice sites. The mechanism by which pre-mRNAs and excised introns are selectively retained within the nucleus is currently unknown. It has been shown if the 5' and 3' splice sites flanking an intron are destroyed, the intron-containing pre-mRNA is exported to the cytoplasm (Chang and Sharp, 1989;Legrain and Rosbash, 1989). Similarly, pre-mRNAs are exported from the nucleus if spliceosome assembly in yeast is disrupted (Legrain and Rosbash, 1989;Ruby and Abelson, 1991). These results have led to a %pliceosome retention" model for the selectivity of RNA export (Green and Zap, 1989), which proposes that pre-mRNAs are retained within the nucleus through their association with spliceosomes. Based upon the apparent immobilization of excised DI introns within the nucleus, we suggest that introns could serve to anchor pre-mRNAs within the nucleus until splicing is completed. The ability of introns to serve as "anchors" could be based on their association with spliceosomes, which may in turn be associated with the nuclear matrix (Verheijen et al., 1988;Carmo-Fonseca et al., 1991). According to this "intron anchor" model, a pre-mRNA that is not spliced cotranscriptionally would be free to be transported toward the nuclear envelope until an intron became assembled with spliceosomal snRNPs. The pre-mRNA would then be immobilized until the intron was excised, after which transport would proceed until another intron became bound by snRNPs. This model is consistent with the reported accumulation of EBV pre-mRNA along nuclear "tracks" that extend from the site of transcription to the nuclear envelope (Lawrence et al., 1989).
The ability to visualize excised Dl introns within the nucleus provides a system to test models relating nuclear structure to RNA processing and export. For example, the "intron anchor" model predicts that introns that are inefficiently recognized by the splicing machinery should be localized at points further along an export pathway than introns that are rapidly excised from a pre-mRNA. This prediction could be tested by introducing mutations near the 5' or 3' splice junctions of a Dl intron and subsequently assaying the nuclear distribution of this intron relative to the other DI introns. Such experiments could provide important insight into the relationships between RNA transcription, RNA splicing, and RNA export as they occur within the highly structured nucleus.