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Evidence that all SC-35 domains contain mRNAs and that transcripts can be structurally constrained within these domains
Journal of
Structural Biology Journal of Structural Biology 140 (2002) 131–139 www.academicpress.com
Evidence that all SC-35 domains contain mRNAs and that transcripts can be structurally constrained within these domains Lindsay S. Shopland, Carol V. Johnson, and Jeanne B. Lawrence* Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Avenue North (S3-138), Worcester, MA 01655-0002, USA Received 14 June 2002, and in revised form 22 August 2002
Abstract A fundamental question of mRNA metabolism concerns the spatial organization of the steps involved in generating mature transcripts and their relationship to SC-35 domains, nuclear compartments enriched in mRNA metabolic factors and poly A+ RNA. Because poly A+ RNA in SC-35 domains remains after transcription inhibition, a prevailing view has been that most or all SC-35 domains do not contain protein-encoding mRNAs but stable RNAs with nuclear functions and thus that these compartments do not have direct roles in mRNA synthesis or transport. However, the transcription, splicing, and transport of transcripts from a specific gene have been shown to occur in association with two of these 15–30 nuclear compartments. Here we show that virtually all SC-35 domains can contain specific mRNAs and that these persist in SC-35 domains after treatment with three different transcription-inhibitory drugs. This suggests perturbation of an mRNA transport step that normally occurs in SC-35 domains and is post-transcriptional but still dependent on ongoing transcription. Finally, even after several hours of transcription arrest, these transcripts do not disperse from SC-35 domains, indicating that they are structurally constrained within them. Our findings importantly suggest a spatially direct role for all SC-35 domains in the coupled steps of mRNA metabolism and transport. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Interchromatin granule cluster; mRNA export; Nuclear compartment; Perichromatin fibril; SFC; snRNP; Speckle
1. Introduction Investigations of mRNA transcription, processing, and export at the molecular level have revealed that many steps in mRNA metabolism are tightly coupled, but it is unclear how these coupled processes are organized spatially within the nucleus. Active transcription has been detected at hundreds of small nuclear sites throughout the nucleus (Jackson et al., 1993; Wansink et al., 1993). In contrast, nuclear poly A+ RNA, which contains both newly synthesized and more mature transcripts, is concentrated in several nuclear sites as well as being dispersed throughout the nucleus at lower levels (Carter et al., 1991; Visa et al., 1993). Interestingly, the concentrated regions of poly A+ RNA correspond to SC-35 domains, the 15–30 largest nuclear ‘‘speckles’’ (0.5–3 lm in diameter). These
*
Corresponding author. Fax: 1-508-856-5178. E-mail address: [email protected] (J.B. Lawrence).
nuclear compartments also contain accumulations of a number of factors required for mRNA synthesis, maturation, and export, including SC-35 and other SR splicing factors, splicing snRNPs, hyperphosphorylated RNA polymerase II, and post-splicing mRNA export factors such as Aly (reviewed in Moen et al., 1995; Reed and Magni, 2001). Do SC-35 domains have a direct or indirect role in nuclear mRNA metabolism? A long-standing view of SC-35 domains is that they do not typically contain mRNA but serve in an indirect capacity as splicing factor storage and recycling centers for active genes throughout the rest of the nucleus (Fakan, 1994; Mattaj, 1994). This is largely based on early RNA pulse-labeling studies using UTP analogs, which reported little if any labeling within SC-35 domains or their ultrastructural equivalents, the interchromatin granule clusters (IGCs) (Fakan et al., 1976; Puvion and Moyne, 1978; Wansink et al., 1993). Moreover, poly A+ RNA remains within SC-35 domains when cells are treated with transcription inhibitors (Lawrence et al., 1993; Huang et al., 1994).
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These domain-associated poly A+ RNAs were hypothesized to be stable transcripts with nuclear function, since typical mRNAs were expected to be exported to the cytoplasm under these conditions (Mattaj, 1994). Consistent with this premise, studies of the dynamics of poly A-containing transcripts and a splicing factor that concentrates in SC-35 domains have indicated largely diffusion-based movement throughout the nucleus, which, for the splicing factor, is not impacted by transcription inhibition (Phair and Misteli, 2000; Politz et al., 1999; Singh et al., 1999). However, a very recent report has indicated that mRNA export may depend on on-going transcription (Griffis et al., 2002), and two other recent studies suggest that nuclear mRNA transport is energy-dependent (Calapez et al., 2002; Kopsky et al., 2002), suggesting that not all transcripts diffuse freely and that their movement may be impacted by transcriptional inhibitors. Other evidence indicates that at least some SC-35 domains are directly involved in mRNA metabolism and transport. In situ hybridization to 21 specific active genes and/or transcripts has shown that half localize consistently at the edges of SC-35 domains (Smith et al., 1999; Xing et al., 1993, 1995). Detailed examination of one of these genes, collagen type 1, a1 (COL1A1), revealed that the majority of its transcripts are spliced near the gene at the domain edge, whereas more mature COL1A1 transcripts accumulate in a post-transcriptional ‘‘track’’ that extends vectorially away from the gene into and throughout the adjacent SC-35 domain (Johnson et al., 2000). These findings may help explain the low levels of uridine-labeled nascent transcripts relative to the high concentrations of poly A+ RNA detected within SC-35 domains, as transcript mass may be greatly reduced by splicing at the domain periphery. Post-transcriptional accumulations of Epstein Barr Virus (EBV) RNA are also unidirectionally transported into an SC-35 domain (Melcak et al., 2000). The movement of certain transcripts into an adjacent SC-35 domain shortly after transcription and splicing suggests that passage through SC-35 domains may be an early step in the transport of specific mRNAs. Importantly, a late-splicing COL1A1 intron has been detected throughout the SC-35 domain, distant from most other introns and the gene. Similarly, a splice-site mutation in COL1A1 that prevents complete splicing results in COL1A1 transcript retention and further accumulation throughout the SC-35 domain (Johnson et al., 2000). Together these findings suggest that all introns must be removed before these transcripts can leave the domain, thus indicating a possible checkpoint in the transport pathway of these (pre)-mRNAs (pre-mRNA and/or fully spliced mRNA (Mattaj, 1994)). Transcripts from most of the endogenous domainassociated genes identified appear restricted to the edge of the SC-35 domain, but domain-filling tracks of car-
diac myosin heavy chain (cMyHC) transcripts, similar to those of COL1A1, have been observed (Smith et al., 1999). Since COL1A1 and cMyHC transcripts are expressed in different cell types, only two of the 15–30 SC35 domains in any given diploid nucleus have been directly shown to contain (pre)-mRNA in their interiors (from two homologs of either gene), though all domains contain poly A+ RNA. This raises the important question of whether SC-35 domains with protein-encoding transcripts in their interiors are the exception or the rule. Because the identity of the poly A+ RNA within SC35 domains has significant implications for both mRNA metabolism and transport. Therefore, we have explored the closely related possibilities that all SC-35 domains typically contain (pre)-mRNA and that export of transcripts from SC-35 domains is coupled to active transcription. The latter possibility has further implications for mechanisms of mRNA transport, since the persistence in SC-35 domains of post-transcriptional (pre)mRNAs normally exported to the cytoplasm would indicate that these transcripts are not necessarily free to diffuse throughout the nucleus.
2. Materials and methods 2.1. Cell culture WI-38 human diploid fibroblasts (ATCC) were grown in EagleÕs basal medium (GibcoBRL) supplemented with 10% heat-inactivated fetal calf serum and 100 units/ml penicillin and streptomycin. Cells were grown at 37 °C on glass coverslips and typically were permeabilized briefly with Triton X-100 and fixed as described (Johnson et al., 1991). Permeabilization removes obscuring cytoplasmic components, including some RNA, but has little effect on nuclear transcripts (Xing and Lawrence, 1991). Cells rapidly and simultaneously fixed and extracted in methanol at )20 °C for 10 min confirmed the distributions of transcripts. To inhibit transcription, cells were treated with 5 lg/ml actinomycin D (ICN) or 40 lg/ml 5,6-dichlorobenzimidazole riboside (DRB) (Calbiochem) for 4 h, or 5 lg/ml a-amanitin (Roche) for 6 h, prior to permeabilization and fixation. To test for general transcriptional activity in nuclei, intact cells were incubated in 2 mM Br-uridine (Research Chemicals) for 40 min. These were rinsed briefly in pre-warmed Hanks and ice-cold 1 PBS before being fixed in 4% paraformaldehyde for 10 min at 4 °C and 10 min at room temperature. They were then permeabilized in 1 PBS, 0.5% Triton X-100 for 15 min prior to immunostaining. 2.2. Fluorescent in situ hybridization (FISH) Hybridizations to RNA and/or DNA were carried out with 50 ng of nick-translated probe labeled with
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either biotin- or digoxigenin-dUTP (Roche) as described (Johnson et al., 1991; Xing et al., 1995). Probes were detected with either TRITC- or FITC-sheep anti-digoxigenin antibody (Roche), FITC–avidin (Roche), or Alexa 596–streptavidin (Molecular Probes). Plasmids for probes were as follows: pHVC1 contains full length COL1A1 cDNA (ATCC), pSTL14 includes full length COL1A2 cDNA (D. Rowe, University of Connecticut Health Center (Lee et al., 1988)), clone 7404 has genomic DNA containing the entire COL1A2 sequence (D. Prockop, MCP–Hahnemann School of Medicine (Korkko et al., 1998)), pHFb-A-1 and LK307 include full length ACTB cDNA and genomic DNA, respectively (J. Stein, University of Massachusetts Medical School, (Ng et al., 1985; Ponte et al., 1984)), and pFH60 contains 4.6 kb (60%) of the complete FN1 cDNA (K. Yamada, NIH, (Obara et al., 1987)). 2.3. Immunostaining SC-35 was detected in nuclei after hybridization as described (Xing et al., 1995), except that cells were stained with 1/200 anti-SC-35 antibody (Sigma) at 37 °C overnight. Incorporated Br-uridine was detected with 1/ 250 anti-Br antibody (Partec) and produced nucleoplasmic but not nucleolar signal in accordance with previous studies (Iborra et al., 1998). This signal was not due to incorporation into DNA, as it was detected without denaturing DNA and was affected by the presence of transcriptional inhibitors (see Section 3). 2.4. Microscopic analysis Cells were examined with a Zeiss Axioplan microscope equipped with a filter wheel, triple-bandpass epifluorescence filter (Chroma Technology), and a 100, 1.4 N.A. objective (Zeiss). Digital images were acquired by either a Photometrics Series 200 CCD or a Photometrics Quantix camera and Metamorph imaging software (Universal Imaging).
3. Results 3.1. Several different (pre)-mRNAs accumulate in the central regions of SC-35 domains Transcripts from most of the specific genes known to associate with SC-35 domains appear to be restricted to the edge of the domain (called a Type II distribution (Smith et al., 1999)). However, the cMyHC and COL1A1 genes produce post-transcriptional accumulations of transcripts within the central regions of domains (Type I). Both COL1A1 and cMyHC are highly expressed and contain multiple introns, characteristics that might play a role in their accumulation within SC-35
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domains. Because the collagen type 1, a2 (COL1A2) shares these characteristics, we examined transcripts from this gene in human fibroblasts to see if they, too, might accumulate within the central regions of SC-35 domains. When COL1A2 transcripts were hybridized with a full-length cDNA probe, they were detected primarily in one or two discrete sites, with weaker, more dispersed signal throughout the nucleoplasm (Fig. 1A, and see below). Scoring >300 of the bright COL1A2 transcript accumulations indicated that 93% contact SC35 domains, with the majority accumulating within the interior portion of the domain in a Type I pattern (Fig. 1B). As previously reported for COL1A1, these COL1A2 transcript accumulations extend from the COL1A2 gene at the domain edge into the domain interior (Figs. 1C–E). That these transcripts are actually in the domainÕs central region was confirmed by 3-D analysis (Shopland et al., submitted). Therefore, COL1A2 is another Type I (pre)-mRNA, and it is expressed with COL1A1 in fibroblast nuclei. Although most COL1A2 RNA signals were within SC-35 domains, a significant fraction (30%) showed a more ‘‘Type II’’ pattern, appearing as small foci restricted to the domain edge. This suggested that the difference between Type I and Type II patterns might be due to lower levels of Type II transcripts within domains that are difficult to detect (Smith et al., 1999; Xing et al., 1995). Alternatively, the prevalence of Type II patterns among domain-associated transcripts might indicate that most transcripts are excluded from the interiors of SC-35 domains. In support of the former possibility, previous analysis of COL1A1 transcripts suggests that different transcripts may have different transit times within SC-35 domains based on the specific kinetics of their metabolism; COL1A1 transcripts containing a remaining late-splicing or splicing-defective intron accumulate to a greater degree within domains (Johnson et al., 2000). We therefore reevaluated the locations of two Type II transcripts, b-actin (ACTB) and fibronectin (FN1), with probes better suited to detecting possible lower levels of transcripts within SC-35 domains. For example, Xing et al. (1995) have demonstrated that signals from already spliced transcripts in the SC-35 domain interior may be missed when a genomic probe is used because it produces a stronger signal at the domain periphery, where most introns may be concentrated (Johnson et al., 2000), whereas a cDNA probe will not favor detection of unspliced transcripts. In addition, the use of fulllength rather than partial cDNA probes also enhances transcript signal. ACTB transcripts had been previously examined with a genomic probe and FN1 with a short partial cDNA (Xing et al., 1993, 1995). The new cDNA ACTB probe not only produced one or two bright transcript signals at the edge of SC-35 domains, but less intense signals could be seen in the interiors of those
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Fig. 1. A significant fraction of SC-35 domains contain (pre)-mRNAs. (A) Two bright COL1A2 (pre-)mRNA accumulations are detected in a WI-38 nucleus with a cDNA probe, as is weaker, more dispersed signal. Signal in the extensive fibroblast cytoplasm is not shown here and is comparatively low due to fixation conditions (see Section 2, Figs. 2L and M). (B) Nuclear COL1A2 transcripts (red) accumulate throughout SC-35 domains (green, insets). Regions of overlap appear yellow or orange. (C–E) A triple-label experiment in which RNA and DNA are discriminated by hybridization conditions and different probe labels (Xing et al., 1995) indicates that COL1A2 RNA (red, detected with a digoxygenin-labeled cDNA probe) extends into an SC-35 domain (blue) from its gene (green, detected with a biotinylated full length genomic probe) at the edge. In the completely merged image (C), regions of red and green overlap appear yellow, and regions of red and blue overlap appear purple or pink. Similar results are observed when labels are reversed, indicating that the more extended RNA signal is not an artifact of the detection method. (F) One ACTB (pre-)mRNA accumulation (green) detected with a cDNA probe extends well into the interior of an SC-35 domain (red) (right, inset); the other is detected only at a domainÕs edge (left). (G–I) ACTB transcripts (green) localize throughout an SC-35 domain (red) in another nucleus. (J–M) FN1 transcript accumulations (red) detected with a 4.6 kb cDNA probe are also found within SC-35 domain central regions (green) (J, inset), though they are occasionally not domain associated (bottom transcript accumulation). (K–M) FN1 transcripts (red) localize throughout an SC-35 domain (grn) in another nucleus. (N, O) Pooled COL1A1 (red), COL1A2 (red), and ACTB (green) cDNA probes show that 6 of 17 SC-35 domains (N (blue), O (white)) contain (pre)-mRNAs in their interiors (arrows and arrowhead), one of which appears small (A, arrowhead) because it is mostly in a different focal plane. Bars, 5 lm.
same domains as well (Figs. 1F–I). This ACTB RNA signal was detected throughout much, though not necessarily all, of the domain and was clearly more intense than the diffuse signal in the rest of the nucleus. That the pattern is largely restricted to two domains indicates that these newly detected signals are specific. Scoring multiple experiments indicates that accumulations of ACTB (pre)-RNA within domains occur in >40% of cells (for 25% of ACTB signals). Use of a longer FN1 cDNA probe (4.6 vs 2 kb (Xing et al., 1993)) also improved the detection of these transcripts, such that 15% of FN1 signals could now be seen to overlap SC35 domains (Figs. 1J–M). Thus, these two Type II transcripts are not excluded from SC-35 domain interiors, as initially appeared, strongly suggesting that the other eight previously examined Type II transcripts are not either.
The cumulative results from our analysis of four specific transcripts (COL1A1, COL1A2, ACTB, and FN1) thus far indicate that the primary accumulations of (pre)-mRNA from these four genes could occupy the interiors of eight SC-35 domains in a single diploid fibroblast nucleus. In a nucleus with only 15–30 domains, this would represent a significant fraction (25–45%) of the domains. To directly demonstrate this point, we hybridized fibroblasts with a mixture of probes to detect ACTB, COL1A1, and COL1A2 RNAs simultaneously, and then stained for SC-35. FN1 probe was omitted because its addition produced background levels that obscured the other signals. Even so, the cell depicted in Fig. 1 (N, O) contains 17 SC-35 domains, 6 of which (35%) contain specific transcripts in their interiors. The fact that transcripts from just three genes label a significant fraction of SC-35 domains strongly predicts
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that most or all SC-35 domains contain (pre)-mRNAs in their interiors. 3.2. Specific (pre)-mRNAs remain in SC-35 domains of transcriptionally inhibited cells The data presented thus far strongly suggest that the poly A+ RNA in many SC-35 domains is composed at least in part of (pre)-mRNAs. If so, then why does poly A+ RNA remain in domains when transcription is inhibited? One possibility is that the transport of at least some (pre)-mRNAs to the cytoplasm is blocked when transcription is inhibited, as has been previously suggested (Brawerman, 1981; Egyhazi, 1974; Herman and Penman, 1977; Lawrence et al., 1989; Sehgal et al., 1976; Vogel and Scholtissek, 1995). This could either be the result of nonspecific effects of the inhibitory drugs used, or, more significantly, because the transcription and export of transcripts may be intimately linked processes. Since an early step in the transport of COL1A1 tran-
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scripts includes a checkpoint in the SC-35 domain interior, where splicing-defective transcripts are retained (Johnson et al., 2000), a perturbation in export might also entrap transcripts there. Therefore, we examined the fate of specific Type I transcripts in cells treated with transcription inhibiting drugs under conditions previously shown to retain poly A+ RNA, to variable degrees, within SC-35 domains (Huang et al., 1994; Lawrence et al., 1993; Visa et al., 1993). When cells inhibited with either actinomycin D or DRB were probed for COL1A1 (pre)-mRNA, bright transcript signals were still observed within SC-35 domains in 75% of nuclei (Figs. 2A, D, and K). These remaining transcripts were detected consistently in multiple experiments and their intensities were not substantially diminished relative to untreated cells, even after 4 h of inhibition (see below). Small particles of COL1A2 transcripts were also present in many inhibited nuclei and associated with several SC-35 domains, though more often at their periphery (Figs. 2B and D).
Fig. 2. Specific (pre-)mRNAs remain within SC-35 domains in transcriptionally inhibited cells. COL1A1 (A, D (red), K) and COL1A2 (B, D (green)) transcripts are readily detected in nuclei after 4 h of DRB (A–D) or actinomycin D (K) inhibition. Most remaining transcripts localize within or at the edges of SC-35 domains (C, and merged images in D), thereby composing a portion of the putative stable nuclear poly A+ RNA. (E–J) A bona fide link between transcription and RNA transport is suggested by 6 hr treatment with the highly specific RNA polymerase II inhibitor, a-amanitin. In both untreated (E, F) and a-amanitin inhibited (G, H) nuclei, COL1A1 transcripts (F, G) are detected at similar levels within SC-35 domains (E, H). Images in (F) and (G) were obtained with identical exposure times for direct comparison of signal intensitites. (I, J) Control experiment verifying that a-amanitin treatment (J) results in fewer, larger, and rounder SC-35 domains (red), and that Br-uridine (green) is not incorporated into nuclear RNA (J), in contrast to transcriptionally active cells (I). (K) Half of the transcriptionally inhibited nuclei contain multiple COL1A1 transcript foci. (L, M) Less intense ‘‘secondary’’ accumulations of COL1A1 (pre)-mRNA (L, M (green)) within SC-35 domains (M, red, see inset) are also frequently observed in normal nuclei and are most concentrated near ‘‘primary’’ transcript accumulations. Bars, 5 lm.
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These did not strictly co-localize with COL1A1 signals and hence were not generated by cross-hybridization. While the abundance of these COL1A2 signals was diminished relative to that of controls, they were still clearly detectable above background. Because actinomycin D and DRB may have additional effects on nuclear factors other than RNA polymerase II, we also examined cells treated with aamanitin, which specifically binds to and inhibits RNA polymerase II (Cochet-Meilhac and Chambon, 1974; Greenleaf et al., 1979). Again, the levels of nuclear COL1A1 transcripts in SC-35 domains were not diminished in cells treated witha-amanitin relative to control cells (Figs. 2E–H).That transcription in these cells was indeed inhibited was confirmed by labeling with Br-uridine prior to fixation (Figs. 2I and J). Cells with the characteristic fewer and larger domains did not incorporate any Br label, yet these still contained COL1A1 transcripts (Figs. 2G and H). Thus, the specific inhibition of RNA polymerase II transcription does not necessarily result in loss of all (pre)-mRNAs from SC-35 domains. Moreover, inhibition does not simply result in all nuclear (pre)-mRNAs accumulating with splicing factors in the more prominent SC-35 domains. Neither FN1 nor ACTB transcripts were detected within domains under these conditions, nor were polyadenylated XIST transcripts, which normally remain associated with the inactive X chromosome and are not exported to the cytoplasm. Further, XIST transcripts that are ectopically expressed and still contain introns ‘‘drift’’ from their chromosome of origin but remain unassociated with SC-35 domains in transcriptionally inhibited cells (Hall and Lawrence, unpublished). In keeping with our analyses of several specific protein-encoding transcripts in normal cells (above), these findings demonstrate directly that the poly A+ RNA that remains in the SC-35 domains of inhibited nuclei includes known proteincoding (pre)-mRNA and is not merely composed of putative stable nuclear poly A+ transcripts that are not (pre)-mRNAs. 3.3. Specific (pre)-mRNAs are immobilized during transcription arrest The experiments above suggest that transcription inhibition impacts the egress of specific protein-encoding transcripts, such as COL1A1, from SC-35 domains. Previous studies have indicated that the mobilities of certain nuclear factors and poly A+ RNAs may be the result of free diffusion (Phair and Misteli, 2000; Politz et al., 1999; Singh et al., 1999) and are accordingly unimpacted by transcription arrest (Phair and Misteli, 2000). In contrast, certain viral transcripts have been found to localize in discrete linear ‘‘tracks’’ that strongly suggest that they are tethered to underlying nuclear structure rather than freely diffusing throughout the
nucleus (Dirks et al., 1995; Lampel et al., 1997; Lawrence et al., 1989). Less distinct tracks have also been observed for some cellular transcripts, including COL1A1, implying that these too might not be free to diffuse. Here we find that COL1A1 and COL1A2 transcripts remain within SC-35 domains in transcriptionally inhibited nuclei and do not disperse evenly throughout the entire nucleus, as would be predicted if they were free to move by diffusion. Hence, our data provide important evidence indicating that these transcripts can be immobilized within a nuclear structure. In further support of the detention of transcripts specifically within SC-35 domains, we found (pre)-mRNAs also accumulated in many other SC-35 domains in about half of the inhibited cells (compare Fig. 2K with Fig. 2A). In these cases, the primary sites of accumulation were not diminished, suggesting that the additional accumulations were instead composed of transcripts previously dispersed throughout the rest of the nucleoplasm. Interestingly, similar but less intense COL1A1 and COL1A2 transcript signals in additional domains were also seen in a significant fraction of uninhibited cells (30%), often those expressing the highest levels of transcripts (Figs. 2L and M; Figs. 1A and B). Because the pattern of these ‘‘secondary’’ accumulations is consistent with a random dispersal of transcripts from the gene into nearby SC-35 domains, with the closest domains accumulating the most transcripts, the transport path for these transcripts appears to include both random diffusion in some nuclear regions and physical constraint in others.
4. Discussion This study provides evidence that the presence of (pre)-mRNA within SC-35 domains is likely the rule rather than the exception; it is not a characteristic of just a few atypical domains. This point is pivotal for understanding the functional significance of these nuclear compartments and the overall spatial organization of (pre)-mRNA metabolism in the nucleus. Our findings support the hypothesis that all SC-35 domains may have direct roles in the transcription, processing, and transport of many (pre)-mRNAs, rather than most domains being reservoirs of factors, devoid of (pre)-mRNAs. Importantly, they also suggest that the transport of specific mRNAs is coupled to active transcription, that this coupling occurs within the SC-35 domain after transcripts have been released from the gene, and that transcripts within the domain are not free to diffuse from them. Several of our observations clearly support that most, if not all, SC-35 domains contain (pre)-mRNA. First, a major fraction (35%) of SC-35 domains were directly shown to contain (pre)-mRNA accumulations in their interior by simultaneous hybridization to transcripts
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from just three genes. Second, improved detection of ACTB and FN1 RNA within SC-35 domains revealed that these ‘‘Type II’’ transcripts do not simply abut storage sites of splicing factors from which they are excluded, but can be detected within the domain interior. This raises the possibility that transcripts from other Type II genes at the edges of SC-35 domains may also pass through these compartments but do not significantly accumulate there. In other studies we have found that transcripts encoding a non-abundant regulatory protein are also enriched within SC-35 domains (Smith et al., in preparation). Hence, this is not only a characteristic of highly abundant transcripts such as those encoding cytoskeletal proteins. Detection of lower levels of COL1A1 and COL1A2 transcripts within many SC-35 domains of normal cells also supports the model that most or all SC-35 domains contain (pre)-mRNAs. This has also been recently observed for p21 transcripts (Hattinger et al., 2002). Because the collective data, including those presented here, indicate that at least half of the known domain-associating genes (5/10) produce transcripts accumulated within SC-35 domains, we predict that the number of different transcripts passing from their genes into SC-35 domains is probably quite large. The final piece of critical evidence is the demonstration that COL1A1 and COL1A2 (pre)-mRNAs remain within SC-35 domains upon transcriptional inhibition. The collective data strongly support the conclusion that the stable poly A+ RNA within all domains is composed, at least in part, of transcripts from mRNA encoding genes. For many years, the predominant view of SC-35 domains has been that they contain little or no (pre)mRNA, an idea that initially arose from e.m. studies of 3 H-uridine incorporation (Fakan, 1994; Mattaj, 1994). Nascent transcripts detected by this method are primarily in perichromatin fibrils (PFs) rather than interchromatin granule clusters (IGCs), which largely correspond to the SC-35 domains seen by light microscopy (Fakan, 1994). However, the relatively low levels of uridine label within SC-35 domains may be explained by recent data indicating that most introns (80% of pre-mRNA mass) have been removed from the COL1A1 transcripts detected within SC-35 domains (Johnson et al., 2000; Moen et al., 1995; Puvion and Puvion-Dutilleul, 1996). While we have not done e.m. here, our findings strongly suggest that IGCs in general contain (pre)-mRNA, and evidence from several other labs agrees with this suggestion (Bridge et al., 1996; Jackson et al., 1993; Melcak et al., 2000; Puvion and PuvionDutilleul, 1996; Wei et al., 1999). The SC-35 domains of cells treated with transcriptional inhibitors are analogous to residual IGCs and not PFs (Fakan, 1994), and we show here that they still contain specific (pre)mRNAs. In addition, our analysis of just a few genes in metabolically active cells indicates that several SC-35
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domains contain specific transcripts in their interiors, and it is highly unlikely that these all correspond strictly to large masses of PFs. As previously suggested, the individual SC-35 domains seen by light microscopy likely correspond at the ultrastructural level to IGCs abutting PFs from specific protein-encoding genes, such that transcription and most splicing take place at the domain edge and the central zones contain high concentrations of largely spliced mRNA and relative excesses of metabolic factors (Johnson et al., 2000; Xing et al., 1995). The data presented here also have important implications for (pre)-mRNA transport. The nuclear retention of COL1A1, and to a lesser extent COL1A2, transcripts was observed after several hours of treatment with three different drugs, each with different mechanisms of inhibition and one that inhibits RNA polymerase II with high specificity (Cochet-Meilhac and Chambon, 1974; Egyhazi, 1974; Yankulov et al., 1995). This strongly suggests a bona fide biological connection between transcription and mRNA trafficking, as opposed to other effects, unrelated to transcriptional arrest. In addition, because the COL1A1 gene is restricted to the SC-35 domain periphery and its transcripts are arrested at a relatively distant point (within the domain), these processes are not coupled simply because polymerases stall on the gene with attached nascent transcripts. Interestingly, recent evidence has shown that the nuclear dynamics of an mRNA export factor strongly depend on active transcription (Griffis et al., 2002), consistent with our findings and the many observations indicating that transcription, splicing, and transport are all coupled to each other (Strasser et al., 2002 and reviewed in Maniatis and Reed, 2002). Because increased accumulation within the SC-35 domain has been observed for splicing defective transcripts, we hypothesize that the domain may constitute a rate-limiting splicing checkpoint that depends on active transcription. Several lines of evidence suggest that domain accumulated transcripts in normal cells are in transit to the cytoplasm and are not necessarily defective transcripts permanently detained in the nucleus. They constitute the vast majority of nuclear COL1A1 transcripts, and it is unlikely that such a large percentage of a geneÕs transcripts would be defective and never leave the nucleus. Moreover, they have much shorter half-lives than known splicing-defective COL1A1 transcripts (Johnson et al., 2000). In addition, error rates should not vary dramatically in transcripts from COL1A1 and COL1A2, which have very similar sequences, yet COL1A1 transcripts are retained significantly more in SC-35 domains upon transcriptional inhibition. Because COL1A1 and COL1A2 are constitutively expressed in fibroblasts, it is difficult to assess the dynamics of their transcripts directly, in situ. However, a very recent study has shown that transcripts from an induced p21 gene transiently
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accumulate within SC-35 domains before collecting in the cytoplasm (Hattinger et al., 2002). Together, these data strongly suggest that transcripts can be exported from SC-35 domains to the cytoplasm. Results from our transcription inhibition experiments also make an important point regarding the fundamental nature of the interaction between mRNA and nuclear structure. We have found that some transcripts within SC-35 domains are not free to diffuse out of them. Further, they suggest that the accumulation of transcripts within domains does not result simply from ‘‘corralling effects’’ imposed by structural constraints on diffusing molecules (e.g., interchromosomal channels) (Politz et al., 1999; Singh et al., 1999; Zachar et al., 1993). Rather, they appear to result from more static interactions with a nuclear structure. This is in contrast to the diffusion-based movement of one of the splicing factors contained in SC-35 domains, as its movement into and out of domains is largely unaffected by transcription inhibitors or reduction in temperature (Kruhlak et al., 2000; Phair and Misteli, 2000). Hence, our studies of inhibited cells suggest that specific normal endogenous transcripts can be detained in the nucleus because of stable interactions with a nuclear structure such as an SC-35 domain, even though some of its components are constantly turning over. While these findings contrast with data indicating diffusion-based movements for the bulk of nuclear poly A+ RNA and specific Chironomus and Drosophila transcripts (Politz et al., 1999; Singh et al., 1999; Zachar et al., 1993), our data do not exclude a role for diffusion in (pre)-mRNA transport. Indeed, the distributions of COL1A1 and COL1A2 ‘‘secondary’’ transcript signals, which are most concentrated near their genes, suggest that these transcripts have dispersed randomly from their primary sites and then pause in a nearby domain. Moreover, the observed differences in the degree to which COL1A1 and COL1A2 transcripts remain in nuclei upon inhibition importantly suggest that there is specificity in transport mechanisms. Further investigation into the status of transcripts that accumulate within SC-35 domains will be key to understanding the role that SC-35 domains play in (pre)-mRNA transport.
Acknowledgments We thank Drs. Kelly Smith, Tom Misteli, and Meg Byron for comments on this manuscript and Drs. David Rowe, Darwin Prockop, Janet Stein, and Kenneth Yamada for clones. This work was supported by National Institutes of Health Grants to J.B.L (GM53234 and GM49254) and Fellowship to L.S.S. (GM18846). The contents are solely the responsibility of the authors and do not necessarily reflect the official views of the NIH.
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Structural Biology Journal of Structural Biology 140 (2002) 131–139 www.academicpress.com
Evidence that all SC-35 domains contain mRNAs and that transcripts can be structurally constrained within these domains Lindsay S. Shopland, Carol V. Johnson, and Jeanne B. Lawrence* Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Avenue North (S3-138), Worcester, MA 01655-0002, USA Received 14 June 2002, and in revised form 22 August 2002
Abstract A fundamental question of mRNA metabolism concerns the spatial organization of the steps involved in generating mature transcripts and their relationship to SC-35 domains, nuclear compartments enriched in mRNA metabolic factors and poly A+ RNA. Because poly A+ RNA in SC-35 domains remains after transcription inhibition, a prevailing view has been that most or all SC-35 domains do not contain protein-encoding mRNAs but stable RNAs with nuclear functions and thus that these compartments do not have direct roles in mRNA synthesis or transport. However, the transcription, splicing, and transport of transcripts from a specific gene have been shown to occur in association with two of these 15–30 nuclear compartments. Here we show that virtually all SC-35 domains can contain specific mRNAs and that these persist in SC-35 domains after treatment with three different transcription-inhibitory drugs. This suggests perturbation of an mRNA transport step that normally occurs in SC-35 domains and is post-transcriptional but still dependent on ongoing transcription. Finally, even after several hours of transcription arrest, these transcripts do not disperse from SC-35 domains, indicating that they are structurally constrained within them. Our findings importantly suggest a spatially direct role for all SC-35 domains in the coupled steps of mRNA metabolism and transport. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Interchromatin granule cluster; mRNA export; Nuclear compartment; Perichromatin fibril; SFC; snRNP; Speckle
1. Introduction Investigations of mRNA transcription, processing, and export at the molecular level have revealed that many steps in mRNA metabolism are tightly coupled, but it is unclear how these coupled processes are organized spatially within the nucleus. Active transcription has been detected at hundreds of small nuclear sites throughout the nucleus (Jackson et al., 1993; Wansink et al., 1993). In contrast, nuclear poly A+ RNA, which contains both newly synthesized and more mature transcripts, is concentrated in several nuclear sites as well as being dispersed throughout the nucleus at lower levels (Carter et al., 1991; Visa et al., 1993). Interestingly, the concentrated regions of poly A+ RNA correspond to SC-35 domains, the 15–30 largest nuclear ‘‘speckles’’ (0.5–3 lm in diameter). These
*
Corresponding author. Fax: 1-508-856-5178. E-mail address: [email protected] (J.B. Lawrence).
nuclear compartments also contain accumulations of a number of factors required for mRNA synthesis, maturation, and export, including SC-35 and other SR splicing factors, splicing snRNPs, hyperphosphorylated RNA polymerase II, and post-splicing mRNA export factors such as Aly (reviewed in Moen et al., 1995; Reed and Magni, 2001). Do SC-35 domains have a direct or indirect role in nuclear mRNA metabolism? A long-standing view of SC-35 domains is that they do not typically contain mRNA but serve in an indirect capacity as splicing factor storage and recycling centers for active genes throughout the rest of the nucleus (Fakan, 1994; Mattaj, 1994). This is largely based on early RNA pulse-labeling studies using UTP analogs, which reported little if any labeling within SC-35 domains or their ultrastructural equivalents, the interchromatin granule clusters (IGCs) (Fakan et al., 1976; Puvion and Moyne, 1978; Wansink et al., 1993). Moreover, poly A+ RNA remains within SC-35 domains when cells are treated with transcription inhibitors (Lawrence et al., 1993; Huang et al., 1994).
1047-8477/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 7 - 8 4 7 7 ( 0 2 ) 0 0 5 0 7 - 5
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These domain-associated poly A+ RNAs were hypothesized to be stable transcripts with nuclear function, since typical mRNAs were expected to be exported to the cytoplasm under these conditions (Mattaj, 1994). Consistent with this premise, studies of the dynamics of poly A-containing transcripts and a splicing factor that concentrates in SC-35 domains have indicated largely diffusion-based movement throughout the nucleus, which, for the splicing factor, is not impacted by transcription inhibition (Phair and Misteli, 2000; Politz et al., 1999; Singh et al., 1999). However, a very recent report has indicated that mRNA export may depend on on-going transcription (Griffis et al., 2002), and two other recent studies suggest that nuclear mRNA transport is energy-dependent (Calapez et al., 2002; Kopsky et al., 2002), suggesting that not all transcripts diffuse freely and that their movement may be impacted by transcriptional inhibitors. Other evidence indicates that at least some SC-35 domains are directly involved in mRNA metabolism and transport. In situ hybridization to 21 specific active genes and/or transcripts has shown that half localize consistently at the edges of SC-35 domains (Smith et al., 1999; Xing et al., 1993, 1995). Detailed examination of one of these genes, collagen type 1, a1 (COL1A1), revealed that the majority of its transcripts are spliced near the gene at the domain edge, whereas more mature COL1A1 transcripts accumulate in a post-transcriptional ‘‘track’’ that extends vectorially away from the gene into and throughout the adjacent SC-35 domain (Johnson et al., 2000). These findings may help explain the low levels of uridine-labeled nascent transcripts relative to the high concentrations of poly A+ RNA detected within SC-35 domains, as transcript mass may be greatly reduced by splicing at the domain periphery. Post-transcriptional accumulations of Epstein Barr Virus (EBV) RNA are also unidirectionally transported into an SC-35 domain (Melcak et al., 2000). The movement of certain transcripts into an adjacent SC-35 domain shortly after transcription and splicing suggests that passage through SC-35 domains may be an early step in the transport of specific mRNAs. Importantly, a late-splicing COL1A1 intron has been detected throughout the SC-35 domain, distant from most other introns and the gene. Similarly, a splice-site mutation in COL1A1 that prevents complete splicing results in COL1A1 transcript retention and further accumulation throughout the SC-35 domain (Johnson et al., 2000). Together these findings suggest that all introns must be removed before these transcripts can leave the domain, thus indicating a possible checkpoint in the transport pathway of these (pre)-mRNAs (pre-mRNA and/or fully spliced mRNA (Mattaj, 1994)). Transcripts from most of the endogenous domainassociated genes identified appear restricted to the edge of the SC-35 domain, but domain-filling tracks of car-
diac myosin heavy chain (cMyHC) transcripts, similar to those of COL1A1, have been observed (Smith et al., 1999). Since COL1A1 and cMyHC transcripts are expressed in different cell types, only two of the 15–30 SC35 domains in any given diploid nucleus have been directly shown to contain (pre)-mRNA in their interiors (from two homologs of either gene), though all domains contain poly A+ RNA. This raises the important question of whether SC-35 domains with protein-encoding transcripts in their interiors are the exception or the rule. Because the identity of the poly A+ RNA within SC35 domains has significant implications for both mRNA metabolism and transport. Therefore, we have explored the closely related possibilities that all SC-35 domains typically contain (pre)-mRNA and that export of transcripts from SC-35 domains is coupled to active transcription. The latter possibility has further implications for mechanisms of mRNA transport, since the persistence in SC-35 domains of post-transcriptional (pre)mRNAs normally exported to the cytoplasm would indicate that these transcripts are not necessarily free to diffuse throughout the nucleus.
2. Materials and methods 2.1. Cell culture WI-38 human diploid fibroblasts (ATCC) were grown in EagleÕs basal medium (GibcoBRL) supplemented with 10% heat-inactivated fetal calf serum and 100 units/ml penicillin and streptomycin. Cells were grown at 37 °C on glass coverslips and typically were permeabilized briefly with Triton X-100 and fixed as described (Johnson et al., 1991). Permeabilization removes obscuring cytoplasmic components, including some RNA, but has little effect on nuclear transcripts (Xing and Lawrence, 1991). Cells rapidly and simultaneously fixed and extracted in methanol at )20 °C for 10 min confirmed the distributions of transcripts. To inhibit transcription, cells were treated with 5 lg/ml actinomycin D (ICN) or 40 lg/ml 5,6-dichlorobenzimidazole riboside (DRB) (Calbiochem) for 4 h, or 5 lg/ml a-amanitin (Roche) for 6 h, prior to permeabilization and fixation. To test for general transcriptional activity in nuclei, intact cells were incubated in 2 mM Br-uridine (Research Chemicals) for 40 min. These were rinsed briefly in pre-warmed Hanks and ice-cold 1 PBS before being fixed in 4% paraformaldehyde for 10 min at 4 °C and 10 min at room temperature. They were then permeabilized in 1 PBS, 0.5% Triton X-100 for 15 min prior to immunostaining. 2.2. Fluorescent in situ hybridization (FISH) Hybridizations to RNA and/or DNA were carried out with 50 ng of nick-translated probe labeled with
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either biotin- or digoxigenin-dUTP (Roche) as described (Johnson et al., 1991; Xing et al., 1995). Probes were detected with either TRITC- or FITC-sheep anti-digoxigenin antibody (Roche), FITC–avidin (Roche), or Alexa 596–streptavidin (Molecular Probes). Plasmids for probes were as follows: pHVC1 contains full length COL1A1 cDNA (ATCC), pSTL14 includes full length COL1A2 cDNA (D. Rowe, University of Connecticut Health Center (Lee et al., 1988)), clone 7404 has genomic DNA containing the entire COL1A2 sequence (D. Prockop, MCP–Hahnemann School of Medicine (Korkko et al., 1998)), pHFb-A-1 and LK307 include full length ACTB cDNA and genomic DNA, respectively (J. Stein, University of Massachusetts Medical School, (Ng et al., 1985; Ponte et al., 1984)), and pFH60 contains 4.6 kb (60%) of the complete FN1 cDNA (K. Yamada, NIH, (Obara et al., 1987)). 2.3. Immunostaining SC-35 was detected in nuclei after hybridization as described (Xing et al., 1995), except that cells were stained with 1/200 anti-SC-35 antibody (Sigma) at 37 °C overnight. Incorporated Br-uridine was detected with 1/ 250 anti-Br antibody (Partec) and produced nucleoplasmic but not nucleolar signal in accordance with previous studies (Iborra et al., 1998). This signal was not due to incorporation into DNA, as it was detected without denaturing DNA and was affected by the presence of transcriptional inhibitors (see Section 3). 2.4. Microscopic analysis Cells were examined with a Zeiss Axioplan microscope equipped with a filter wheel, triple-bandpass epifluorescence filter (Chroma Technology), and a 100, 1.4 N.A. objective (Zeiss). Digital images were acquired by either a Photometrics Series 200 CCD or a Photometrics Quantix camera and Metamorph imaging software (Universal Imaging).
3. Results 3.1. Several different (pre)-mRNAs accumulate in the central regions of SC-35 domains Transcripts from most of the specific genes known to associate with SC-35 domains appear to be restricted to the edge of the domain (called a Type II distribution (Smith et al., 1999)). However, the cMyHC and COL1A1 genes produce post-transcriptional accumulations of transcripts within the central regions of domains (Type I). Both COL1A1 and cMyHC are highly expressed and contain multiple introns, characteristics that might play a role in their accumulation within SC-35
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domains. Because the collagen type 1, a2 (COL1A2) shares these characteristics, we examined transcripts from this gene in human fibroblasts to see if they, too, might accumulate within the central regions of SC-35 domains. When COL1A2 transcripts were hybridized with a full-length cDNA probe, they were detected primarily in one or two discrete sites, with weaker, more dispersed signal throughout the nucleoplasm (Fig. 1A, and see below). Scoring >300 of the bright COL1A2 transcript accumulations indicated that 93% contact SC35 domains, with the majority accumulating within the interior portion of the domain in a Type I pattern (Fig. 1B). As previously reported for COL1A1, these COL1A2 transcript accumulations extend from the COL1A2 gene at the domain edge into the domain interior (Figs. 1C–E). That these transcripts are actually in the domainÕs central region was confirmed by 3-D analysis (Shopland et al., submitted). Therefore, COL1A2 is another Type I (pre)-mRNA, and it is expressed with COL1A1 in fibroblast nuclei. Although most COL1A2 RNA signals were within SC-35 domains, a significant fraction (30%) showed a more ‘‘Type II’’ pattern, appearing as small foci restricted to the domain edge. This suggested that the difference between Type I and Type II patterns might be due to lower levels of Type II transcripts within domains that are difficult to detect (Smith et al., 1999; Xing et al., 1995). Alternatively, the prevalence of Type II patterns among domain-associated transcripts might indicate that most transcripts are excluded from the interiors of SC-35 domains. In support of the former possibility, previous analysis of COL1A1 transcripts suggests that different transcripts may have different transit times within SC-35 domains based on the specific kinetics of their metabolism; COL1A1 transcripts containing a remaining late-splicing or splicing-defective intron accumulate to a greater degree within domains (Johnson et al., 2000). We therefore reevaluated the locations of two Type II transcripts, b-actin (ACTB) and fibronectin (FN1), with probes better suited to detecting possible lower levels of transcripts within SC-35 domains. For example, Xing et al. (1995) have demonstrated that signals from already spliced transcripts in the SC-35 domain interior may be missed when a genomic probe is used because it produces a stronger signal at the domain periphery, where most introns may be concentrated (Johnson et al., 2000), whereas a cDNA probe will not favor detection of unspliced transcripts. In addition, the use of fulllength rather than partial cDNA probes also enhances transcript signal. ACTB transcripts had been previously examined with a genomic probe and FN1 with a short partial cDNA (Xing et al., 1993, 1995). The new cDNA ACTB probe not only produced one or two bright transcript signals at the edge of SC-35 domains, but less intense signals could be seen in the interiors of those
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Fig. 1. A significant fraction of SC-35 domains contain (pre)-mRNAs. (A) Two bright COL1A2 (pre-)mRNA accumulations are detected in a WI-38 nucleus with a cDNA probe, as is weaker, more dispersed signal. Signal in the extensive fibroblast cytoplasm is not shown here and is comparatively low due to fixation conditions (see Section 2, Figs. 2L and M). (B) Nuclear COL1A2 transcripts (red) accumulate throughout SC-35 domains (green, insets). Regions of overlap appear yellow or orange. (C–E) A triple-label experiment in which RNA and DNA are discriminated by hybridization conditions and different probe labels (Xing et al., 1995) indicates that COL1A2 RNA (red, detected with a digoxygenin-labeled cDNA probe) extends into an SC-35 domain (blue) from its gene (green, detected with a biotinylated full length genomic probe) at the edge. In the completely merged image (C), regions of red and green overlap appear yellow, and regions of red and blue overlap appear purple or pink. Similar results are observed when labels are reversed, indicating that the more extended RNA signal is not an artifact of the detection method. (F) One ACTB (pre-)mRNA accumulation (green) detected with a cDNA probe extends well into the interior of an SC-35 domain (red) (right, inset); the other is detected only at a domainÕs edge (left). (G–I) ACTB transcripts (green) localize throughout an SC-35 domain (red) in another nucleus. (J–M) FN1 transcript accumulations (red) detected with a 4.6 kb cDNA probe are also found within SC-35 domain central regions (green) (J, inset), though they are occasionally not domain associated (bottom transcript accumulation). (K–M) FN1 transcripts (red) localize throughout an SC-35 domain (grn) in another nucleus. (N, O) Pooled COL1A1 (red), COL1A2 (red), and ACTB (green) cDNA probes show that 6 of 17 SC-35 domains (N (blue), O (white)) contain (pre)-mRNAs in their interiors (arrows and arrowhead), one of which appears small (A, arrowhead) because it is mostly in a different focal plane. Bars, 5 lm.
same domains as well (Figs. 1F–I). This ACTB RNA signal was detected throughout much, though not necessarily all, of the domain and was clearly more intense than the diffuse signal in the rest of the nucleus. That the pattern is largely restricted to two domains indicates that these newly detected signals are specific. Scoring multiple experiments indicates that accumulations of ACTB (pre)-RNA within domains occur in >40% of cells (for 25% of ACTB signals). Use of a longer FN1 cDNA probe (4.6 vs 2 kb (Xing et al., 1993)) also improved the detection of these transcripts, such that 15% of FN1 signals could now be seen to overlap SC35 domains (Figs. 1J–M). Thus, these two Type II transcripts are not excluded from SC-35 domain interiors, as initially appeared, strongly suggesting that the other eight previously examined Type II transcripts are not either.
The cumulative results from our analysis of four specific transcripts (COL1A1, COL1A2, ACTB, and FN1) thus far indicate that the primary accumulations of (pre)-mRNA from these four genes could occupy the interiors of eight SC-35 domains in a single diploid fibroblast nucleus. In a nucleus with only 15–30 domains, this would represent a significant fraction (25–45%) of the domains. To directly demonstrate this point, we hybridized fibroblasts with a mixture of probes to detect ACTB, COL1A1, and COL1A2 RNAs simultaneously, and then stained for SC-35. FN1 probe was omitted because its addition produced background levels that obscured the other signals. Even so, the cell depicted in Fig. 1 (N, O) contains 17 SC-35 domains, 6 of which (35%) contain specific transcripts in their interiors. The fact that transcripts from just three genes label a significant fraction of SC-35 domains strongly predicts
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that most or all SC-35 domains contain (pre)-mRNAs in their interiors. 3.2. Specific (pre)-mRNAs remain in SC-35 domains of transcriptionally inhibited cells The data presented thus far strongly suggest that the poly A+ RNA in many SC-35 domains is composed at least in part of (pre)-mRNAs. If so, then why does poly A+ RNA remain in domains when transcription is inhibited? One possibility is that the transport of at least some (pre)-mRNAs to the cytoplasm is blocked when transcription is inhibited, as has been previously suggested (Brawerman, 1981; Egyhazi, 1974; Herman and Penman, 1977; Lawrence et al., 1989; Sehgal et al., 1976; Vogel and Scholtissek, 1995). This could either be the result of nonspecific effects of the inhibitory drugs used, or, more significantly, because the transcription and export of transcripts may be intimately linked processes. Since an early step in the transport of COL1A1 tran-
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scripts includes a checkpoint in the SC-35 domain interior, where splicing-defective transcripts are retained (Johnson et al., 2000), a perturbation in export might also entrap transcripts there. Therefore, we examined the fate of specific Type I transcripts in cells treated with transcription inhibiting drugs under conditions previously shown to retain poly A+ RNA, to variable degrees, within SC-35 domains (Huang et al., 1994; Lawrence et al., 1993; Visa et al., 1993). When cells inhibited with either actinomycin D or DRB were probed for COL1A1 (pre)-mRNA, bright transcript signals were still observed within SC-35 domains in 75% of nuclei (Figs. 2A, D, and K). These remaining transcripts were detected consistently in multiple experiments and their intensities were not substantially diminished relative to untreated cells, even after 4 h of inhibition (see below). Small particles of COL1A2 transcripts were also present in many inhibited nuclei and associated with several SC-35 domains, though more often at their periphery (Figs. 2B and D).
Fig. 2. Specific (pre-)mRNAs remain within SC-35 domains in transcriptionally inhibited cells. COL1A1 (A, D (red), K) and COL1A2 (B, D (green)) transcripts are readily detected in nuclei after 4 h of DRB (A–D) or actinomycin D (K) inhibition. Most remaining transcripts localize within or at the edges of SC-35 domains (C, and merged images in D), thereby composing a portion of the putative stable nuclear poly A+ RNA. (E–J) A bona fide link between transcription and RNA transport is suggested by 6 hr treatment with the highly specific RNA polymerase II inhibitor, a-amanitin. In both untreated (E, F) and a-amanitin inhibited (G, H) nuclei, COL1A1 transcripts (F, G) are detected at similar levels within SC-35 domains (E, H). Images in (F) and (G) were obtained with identical exposure times for direct comparison of signal intensitites. (I, J) Control experiment verifying that a-amanitin treatment (J) results in fewer, larger, and rounder SC-35 domains (red), and that Br-uridine (green) is not incorporated into nuclear RNA (J), in contrast to transcriptionally active cells (I). (K) Half of the transcriptionally inhibited nuclei contain multiple COL1A1 transcript foci. (L, M) Less intense ‘‘secondary’’ accumulations of COL1A1 (pre)-mRNA (L, M (green)) within SC-35 domains (M, red, see inset) are also frequently observed in normal nuclei and are most concentrated near ‘‘primary’’ transcript accumulations. Bars, 5 lm.
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These did not strictly co-localize with COL1A1 signals and hence were not generated by cross-hybridization. While the abundance of these COL1A2 signals was diminished relative to that of controls, they were still clearly detectable above background. Because actinomycin D and DRB may have additional effects on nuclear factors other than RNA polymerase II, we also examined cells treated with aamanitin, which specifically binds to and inhibits RNA polymerase II (Cochet-Meilhac and Chambon, 1974; Greenleaf et al., 1979). Again, the levels of nuclear COL1A1 transcripts in SC-35 domains were not diminished in cells treated witha-amanitin relative to control cells (Figs. 2E–H).That transcription in these cells was indeed inhibited was confirmed by labeling with Br-uridine prior to fixation (Figs. 2I and J). Cells with the characteristic fewer and larger domains did not incorporate any Br label, yet these still contained COL1A1 transcripts (Figs. 2G and H). Thus, the specific inhibition of RNA polymerase II transcription does not necessarily result in loss of all (pre)-mRNAs from SC-35 domains. Moreover, inhibition does not simply result in all nuclear (pre)-mRNAs accumulating with splicing factors in the more prominent SC-35 domains. Neither FN1 nor ACTB transcripts were detected within domains under these conditions, nor were polyadenylated XIST transcripts, which normally remain associated with the inactive X chromosome and are not exported to the cytoplasm. Further, XIST transcripts that are ectopically expressed and still contain introns ‘‘drift’’ from their chromosome of origin but remain unassociated with SC-35 domains in transcriptionally inhibited cells (Hall and Lawrence, unpublished). In keeping with our analyses of several specific protein-encoding transcripts in normal cells (above), these findings demonstrate directly that the poly A+ RNA that remains in the SC-35 domains of inhibited nuclei includes known proteincoding (pre)-mRNA and is not merely composed of putative stable nuclear poly A+ transcripts that are not (pre)-mRNAs. 3.3. Specific (pre)-mRNAs are immobilized during transcription arrest The experiments above suggest that transcription inhibition impacts the egress of specific protein-encoding transcripts, such as COL1A1, from SC-35 domains. Previous studies have indicated that the mobilities of certain nuclear factors and poly A+ RNAs may be the result of free diffusion (Phair and Misteli, 2000; Politz et al., 1999; Singh et al., 1999) and are accordingly unimpacted by transcription arrest (Phair and Misteli, 2000). In contrast, certain viral transcripts have been found to localize in discrete linear ‘‘tracks’’ that strongly suggest that they are tethered to underlying nuclear structure rather than freely diffusing throughout the
nucleus (Dirks et al., 1995; Lampel et al., 1997; Lawrence et al., 1989). Less distinct tracks have also been observed for some cellular transcripts, including COL1A1, implying that these too might not be free to diffuse. Here we find that COL1A1 and COL1A2 transcripts remain within SC-35 domains in transcriptionally inhibited nuclei and do not disperse evenly throughout the entire nucleus, as would be predicted if they were free to move by diffusion. Hence, our data provide important evidence indicating that these transcripts can be immobilized within a nuclear structure. In further support of the detention of transcripts specifically within SC-35 domains, we found (pre)-mRNAs also accumulated in many other SC-35 domains in about half of the inhibited cells (compare Fig. 2K with Fig. 2A). In these cases, the primary sites of accumulation were not diminished, suggesting that the additional accumulations were instead composed of transcripts previously dispersed throughout the rest of the nucleoplasm. Interestingly, similar but less intense COL1A1 and COL1A2 transcript signals in additional domains were also seen in a significant fraction of uninhibited cells (30%), often those expressing the highest levels of transcripts (Figs. 2L and M; Figs. 1A and B). Because the pattern of these ‘‘secondary’’ accumulations is consistent with a random dispersal of transcripts from the gene into nearby SC-35 domains, with the closest domains accumulating the most transcripts, the transport path for these transcripts appears to include both random diffusion in some nuclear regions and physical constraint in others.
4. Discussion This study provides evidence that the presence of (pre)-mRNA within SC-35 domains is likely the rule rather than the exception; it is not a characteristic of just a few atypical domains. This point is pivotal for understanding the functional significance of these nuclear compartments and the overall spatial organization of (pre)-mRNA metabolism in the nucleus. Our findings support the hypothesis that all SC-35 domains may have direct roles in the transcription, processing, and transport of many (pre)-mRNAs, rather than most domains being reservoirs of factors, devoid of (pre)-mRNAs. Importantly, they also suggest that the transport of specific mRNAs is coupled to active transcription, that this coupling occurs within the SC-35 domain after transcripts have been released from the gene, and that transcripts within the domain are not free to diffuse from them. Several of our observations clearly support that most, if not all, SC-35 domains contain (pre)-mRNA. First, a major fraction (35%) of SC-35 domains were directly shown to contain (pre)-mRNA accumulations in their interior by simultaneous hybridization to transcripts
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from just three genes. Second, improved detection of ACTB and FN1 RNA within SC-35 domains revealed that these ‘‘Type II’’ transcripts do not simply abut storage sites of splicing factors from which they are excluded, but can be detected within the domain interior. This raises the possibility that transcripts from other Type II genes at the edges of SC-35 domains may also pass through these compartments but do not significantly accumulate there. In other studies we have found that transcripts encoding a non-abundant regulatory protein are also enriched within SC-35 domains (Smith et al., in preparation). Hence, this is not only a characteristic of highly abundant transcripts such as those encoding cytoskeletal proteins. Detection of lower levels of COL1A1 and COL1A2 transcripts within many SC-35 domains of normal cells also supports the model that most or all SC-35 domains contain (pre)-mRNAs. This has also been recently observed for p21 transcripts (Hattinger et al., 2002). Because the collective data, including those presented here, indicate that at least half of the known domain-associating genes (5/10) produce transcripts accumulated within SC-35 domains, we predict that the number of different transcripts passing from their genes into SC-35 domains is probably quite large. The final piece of critical evidence is the demonstration that COL1A1 and COL1A2 (pre)-mRNAs remain within SC-35 domains upon transcriptional inhibition. The collective data strongly support the conclusion that the stable poly A+ RNA within all domains is composed, at least in part, of transcripts from mRNA encoding genes. For many years, the predominant view of SC-35 domains has been that they contain little or no (pre)mRNA, an idea that initially arose from e.m. studies of 3 H-uridine incorporation (Fakan, 1994; Mattaj, 1994). Nascent transcripts detected by this method are primarily in perichromatin fibrils (PFs) rather than interchromatin granule clusters (IGCs), which largely correspond to the SC-35 domains seen by light microscopy (Fakan, 1994). However, the relatively low levels of uridine label within SC-35 domains may be explained by recent data indicating that most introns (80% of pre-mRNA mass) have been removed from the COL1A1 transcripts detected within SC-35 domains (Johnson et al., 2000; Moen et al., 1995; Puvion and Puvion-Dutilleul, 1996). While we have not done e.m. here, our findings strongly suggest that IGCs in general contain (pre)-mRNA, and evidence from several other labs agrees with this suggestion (Bridge et al., 1996; Jackson et al., 1993; Melcak et al., 2000; Puvion and PuvionDutilleul, 1996; Wei et al., 1999). The SC-35 domains of cells treated with transcriptional inhibitors are analogous to residual IGCs and not PFs (Fakan, 1994), and we show here that they still contain specific (pre)mRNAs. In addition, our analysis of just a few genes in metabolically active cells indicates that several SC-35
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domains contain specific transcripts in their interiors, and it is highly unlikely that these all correspond strictly to large masses of PFs. As previously suggested, the individual SC-35 domains seen by light microscopy likely correspond at the ultrastructural level to IGCs abutting PFs from specific protein-encoding genes, such that transcription and most splicing take place at the domain edge and the central zones contain high concentrations of largely spliced mRNA and relative excesses of metabolic factors (Johnson et al., 2000; Xing et al., 1995). The data presented here also have important implications for (pre)-mRNA transport. The nuclear retention of COL1A1, and to a lesser extent COL1A2, transcripts was observed after several hours of treatment with three different drugs, each with different mechanisms of inhibition and one that inhibits RNA polymerase II with high specificity (Cochet-Meilhac and Chambon, 1974; Egyhazi, 1974; Yankulov et al., 1995). This strongly suggests a bona fide biological connection between transcription and mRNA trafficking, as opposed to other effects, unrelated to transcriptional arrest. In addition, because the COL1A1 gene is restricted to the SC-35 domain periphery and its transcripts are arrested at a relatively distant point (within the domain), these processes are not coupled simply because polymerases stall on the gene with attached nascent transcripts. Interestingly, recent evidence has shown that the nuclear dynamics of an mRNA export factor strongly depend on active transcription (Griffis et al., 2002), consistent with our findings and the many observations indicating that transcription, splicing, and transport are all coupled to each other (Strasser et al., 2002 and reviewed in Maniatis and Reed, 2002). Because increased accumulation within the SC-35 domain has been observed for splicing defective transcripts, we hypothesize that the domain may constitute a rate-limiting splicing checkpoint that depends on active transcription. Several lines of evidence suggest that domain accumulated transcripts in normal cells are in transit to the cytoplasm and are not necessarily defective transcripts permanently detained in the nucleus. They constitute the vast majority of nuclear COL1A1 transcripts, and it is unlikely that such a large percentage of a geneÕs transcripts would be defective and never leave the nucleus. Moreover, they have much shorter half-lives than known splicing-defective COL1A1 transcripts (Johnson et al., 2000). In addition, error rates should not vary dramatically in transcripts from COL1A1 and COL1A2, which have very similar sequences, yet COL1A1 transcripts are retained significantly more in SC-35 domains upon transcriptional inhibition. Because COL1A1 and COL1A2 are constitutively expressed in fibroblasts, it is difficult to assess the dynamics of their transcripts directly, in situ. However, a very recent study has shown that transcripts from an induced p21 gene transiently
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accumulate within SC-35 domains before collecting in the cytoplasm (Hattinger et al., 2002). Together, these data strongly suggest that transcripts can be exported from SC-35 domains to the cytoplasm. Results from our transcription inhibition experiments also make an important point regarding the fundamental nature of the interaction between mRNA and nuclear structure. We have found that some transcripts within SC-35 domains are not free to diffuse out of them. Further, they suggest that the accumulation of transcripts within domains does not result simply from ‘‘corralling effects’’ imposed by structural constraints on diffusing molecules (e.g., interchromosomal channels) (Politz et al., 1999; Singh et al., 1999; Zachar et al., 1993). Rather, they appear to result from more static interactions with a nuclear structure. This is in contrast to the diffusion-based movement of one of the splicing factors contained in SC-35 domains, as its movement into and out of domains is largely unaffected by transcription inhibitors or reduction in temperature (Kruhlak et al., 2000; Phair and Misteli, 2000). Hence, our studies of inhibited cells suggest that specific normal endogenous transcripts can be detained in the nucleus because of stable interactions with a nuclear structure such as an SC-35 domain, even though some of its components are constantly turning over. While these findings contrast with data indicating diffusion-based movements for the bulk of nuclear poly A+ RNA and specific Chironomus and Drosophila transcripts (Politz et al., 1999; Singh et al., 1999; Zachar et al., 1993), our data do not exclude a role for diffusion in (pre)-mRNA transport. Indeed, the distributions of COL1A1 and COL1A2 ‘‘secondary’’ transcript signals, which are most concentrated near their genes, suggest that these transcripts have dispersed randomly from their primary sites and then pause in a nearby domain. Moreover, the observed differences in the degree to which COL1A1 and COL1A2 transcripts remain in nuclei upon inhibition importantly suggest that there is specificity in transport mechanisms. Further investigation into the status of transcripts that accumulate within SC-35 domains will be key to understanding the role that SC-35 domains play in (pre)-mRNA transport.
Acknowledgments We thank Drs. Kelly Smith, Tom Misteli, and Meg Byron for comments on this manuscript and Drs. David Rowe, Darwin Prockop, Janet Stein, and Kenneth Yamada for clones. This work was supported by National Institutes of Health Grants to J.B.L (GM53234 and GM49254) and Fellowship to L.S.S. (GM18846). The contents are solely the responsibility of the authors and do not necessarily reflect the official views of the NIH.
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