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• W2066016774 abstract "Article15 March 1997free access Dynamic relocation of transcription and splicing factors dependent upon transcriptional activity Changqing Zeng Changqing Zeng Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030 USA Search for more papers by this author Euikyung Kim Euikyung Kim Department of Pathology, Yale University School of Medicine, New Haven, CT, 06510 USA Search for more papers by this author Stephen L. Warren Stephen L. Warren Department of Pathology, Yale University School of Medicine, New Haven, CT, 06510 USA Search for more papers by this author Susan M. Berget Corresponding Author Susan M. Berget Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030 USA Search for more papers by this author Changqing Zeng Changqing Zeng Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030 USA Search for more papers by this author Euikyung Kim Euikyung Kim Department of Pathology, Yale University School of Medicine, New Haven, CT, 06510 USA Search for more papers by this author Stephen L. Warren Stephen L. Warren Department of Pathology, Yale University School of Medicine, New Haven, CT, 06510 USA Search for more papers by this author Susan M. Berget Corresponding Author Susan M. Berget Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030 USA Search for more papers by this author Author Information Changqing Zeng1, Euikyung Kim2, Stephen L. Warren2 and Susan M. Berget 1 1Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030 USA 2Department of Pathology, Yale University School of Medicine, New Haven, CT, 06510 USA The EMBO Journal (1997)16:1401-1412https://doi.org/10.1093/emboj/16.6.1401 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Recent interest in understanding the spatial organization of gene expression has focused attention on nuclear structures known as speckles or interchromatin granule clusters (IGCs) revealed by immunofluorescence or electron microscopy. Staining of nuclear factors involved in pre-mRNA splicing or, more recently, transcription, reveals 20–40 speckles per nucleus, resulting in the intriguing suggestion that speckles are nuclear sites of transcription and processing. In contrast, other investigations have observed transcription in other areas of the nucleus. In this study, we have examined the localization of active transcription as detected by uridine incorporation and recently developed RNA polymerase II antibodies, and compared this pattern with that of known splicing and polyadenylation factors. Our results indicate that in actively transcribing cells, transcription and splicing factors are dispersed throughout the nucleus with abundant sites of preferred localization. In contrast, in poorly transcribing cells, polymerase II and splicing factors localize to speckles. In nuclei inactivated for transcription by drugs or heat shock, the speckle type of co-localization is accentuated. These observations suggest that bulk transcription and splicing occur throughout the nucleus during periods of active transcription; and that factors involved in these two processes re-locate to minimal speckle domains during periods of inactive transcription. Introduction In recent years, a number of studies have addressed the spatial organization of gene expression within mammalian nuclei. Key to this endeavor have been experiments localizing polymerase II-derived nascent transcripts and the factors that transcribe, splice and polyadenylate these RNAs. A number of antibodies specific for elements of the transcription or pre-mRNA processing machinery have been used to reveal subnuclear structures associated with pre-mRNA (reviewed in Spector, 1993; Fakan, 1994; Moen, et al., 1995; van Driel et al., 1995). One particularly striking staining pattern is that observed using an antibody raised against the conserved splicing factor, SC35. This antibody brightly stains nuclei from a wide variety of tissue culture cells in a pattern of localized staining termed ‘speckles’ (Fu and Maniatis, 1990; Spector et al., 1991). Other splicing-related factors were also localized to speckles (Spector, 1984, 1990; Nyman et al., 1986; Carmo-Fonseca et al., 1992; Huang and Spector, 1992; Krause et al., 1994; for reviews, see Spector, 1993; van Driel et al., 1995). Cultured mammalian cells commonly contain 20–40 speckles, raising the intriguing possibility that RNA processing is restricted to minimal domains within mammalian nuclei. Such a hypothesis immediately suggested either that many pre-mRNAs should be synthesized within the vicinity of speckles or that nascent pre-mRNAs must be transported to the speckles for processing. Experiments to address the possibility that speckles represent sites of transcription or processing of pre-mRNA have utilized localization of either bulk or individual transcripts. Use of oligo(dT) probes to detect bulk polyadenylated RNA revealed a speckled localization (Carter et al., 1991, 1993). Several identified unique pre-mRNAs have also been localized to speckles defined by staining with the anti-SC35 antibody (Huang and Spector, 1991; Lawrence et al., 1993; Xing et al., 1993, 1995; reviewed in Moen et al., 1995). Speckled localization was especially prominent for microinjected RNAs or transcripts produced from transfected DNAs (Wang et al., 1991; Huang and Spector, 1996). Such data suggested that the speckled pattern reflected localized accumulation of active splicing and transcription factors. An alternative proposal has suggested speckled nuclear structures as storage sites of splicing and transcription factors distal from the sites of bulk transcription occurring throughout the nucleoplasm and associated with chromatin (reviewed in Spector, 1993; van Driel et al., 1995). Supporting evidence for this proposal came from early electron microscopy studies in which bulk transcription was localized by short pulses of [3H]uridine incorporation. Nascent RNA was detected within the perichromatin fibrils that reside between the interchromatin granule clusters (IGCs) with little accumulation at the IGCs, or speckles (reviewed in Fakan and Puvion, 1980; Fakan, 1994; Hendzel and Bazett-Jones, 1995). Subsequent in situ nucleotide incorporation experiments using fluorescence microscopy have confirmed this observation via the detection of transcription at many sites within the nucleus other than the speckles (Jackson et al., 1993; Wansink et al., 1993). Furthermore, multiple identified pre-mRNAs have been localized to regions of the nucleoplasm between the speckles (Zhang et al., 1994). These results raised questions about speckles being the sites of pre-mRNA generation and subsequent splicing. Such incorporation studies, however, are compromised by the inherent difficulty in ascribing bulk transcription to pol II-driven pre-mRNA synthesis (Moen et al., 1995). Determining the function in transcription and splicing of speckles is an important issue because of the ramifications that alternate interpretations have on the spatial relationship between transcription and RNA processing within the nucleus. If transcription and processing are coupled, as studies in Drosophila indicate (Beyer et al., 1988), then it is difficult to visualize how all of the active transcription sites in the average nucleus can be effectively coupled to 20–40 speckles containing processing factors. It also becomes difficult to explain why so much transcription occurs distal to speckles when in situ incorporation experiments are performed. If transcription and splicing are spatially uncoupled within the nucleus, it is difficult to envisage how extremely large pre-mRNAs can be made as a unit and then transported within the nucleus to speckle-type processing centers. Thus, it is difficult to reconcile observations that indicate disperse transcription centers and localized processing centers. Given the striking visual observation that many factors involved in splicing can be found in speckles, it becomes imperative to ascertain if speckles are indeed the sites of active pre-mRNA production and/or processing. Recently, two antibodies directed toward RNA polymerase II were developed (Bregman et al., 1995). One of these antibodies recognizes pol II hyperphosphorylated within the C-terminal domain (CTD) of the large subunit, whereas the other recognizes hyper-, hypo- and intermediately phosphorylated forms of the CTD. In this study, we show that both pol II antibodies can recognize transcriptionally engaged polymerase molecules, indicating that both antibodies can be used to detect sites of active transcription within the nuclei of mammalian cells. Using these antibodies and other antibodies specific to RNA processing factors, we have observed that transcription and RNA processing overlap within a broad meshwork occupying the non-nucleolar portion of the nucleus with hundreds of ‘dots’ of preferred localization overlaying the network. Observation of the meshwork was dependent on the ability to detect active transcription by bromo-uridine triphosphate (BrU) incorporation. In cells in which BrU incorporation was abundant, BrU incorporation, the large subunit of polymerase II, and splicing and polyadenylation factors were broadly located within the nucleoplasm and did not concentrate at speckles. In contrast, cells that poorly incorporated BrU demonstrated a strikingly different pattern in that both pol II antibodies and splicing factor antibodies co-localized to classic speckles, suggesting a redistribution of transcription and splicing factors during periods of inactive transcription. A similar speckled localization of pol II was observed when transcription was experimentally arrested with either drugs or heat shock, supporting such a redistribution. Therefore, our data suggest that active transcription and splicing occur throughout the nucleoplasm. Furthermore, they support an alternative definition of speckles as dynamic locales within the nucleoplasm in which transcription and processing factors localize between rounds of active pre-mRNA synthesis, perhaps for some form of recycling and reactivation. Our data also suggest that very active transcripts, such as those previously localized to speckles, may be found preferentially in, or adjacent to, speckles because they provide a local high concentration of processing factors following recycling. Results Monoclonal antibodies H5 and H14 recognize transcriptionally engaged polymerase II molecules Monoclonal antibodies reactive for phosphorylated forms of mammalian polymerase II have recently been developed (Bregman et al., 1995). One of these antibodies, mAb H5, is specific for a hyperphosphorylated form of the large subunit of pol II (IIo); a second antibody, mAb H14, recognizes various forms of the phosphorylated pol II large subunit. A population of non-transcribing polymerase molecules that are phosphorylated on the CTD has been detected by both H5 and H14 (Bregman et al., 1995; Kim et al., 1997). However, phosphorylation of the CTD takes place concomitant with the initiation of transcription (reviewed by Dahmus, 1995); consequently, we asked if mAb H5 and H14 recognize phospho-epitopes on actively transcribing polymerase molecules. For this purpose, we used an in situ photoaffinity labeling technique (Bartholomew et al., 1986). Briefly, nuclear run-on transcription was performed with isolated HeLa cell nuclei in the presence of [α-32P]CTP (labeling nucleotide), 4-thio-UTP (photoactive RNA–protein cross-linking nucleotide) and 3′-O-methyl GTP (chain-terminating nucleotide). Then nuclei were exposed to UV light (λmax = 310 nm) to induce covalent cross-linking of the radiolabeled RNAs to proteins. The nuclei were extracted after digestion with RNase to trim excess RNA from the proteins, and the extract was used immediately for immunoprecipitation with mAbs H14 and H5. The results indicated that, in the absence of the transcription inhibitor α-amanitin, mAbs H5 and H14 immunoprecipitated three distinct [α-32P]CTP-radiolabeled polymerase II subunits: IIo (∼240 kDa), IIa (∼220 kDa) and IIc (140 kDa) (Figure 1A). mAb H5, which recognizes a hyperphosphorylated form(s) of polymerase II (Bregman et al., 1995), immunoprecipitated predominantly [α–32P]CTP-radiolabeled Pol IIo. Trace [α-32P]CTP-radiolabeled Pol IIa was also present (Figure 1A, lane 1). mAb H14, which recognizes multiple phosphorylated forms of polymerase, immunoprecipitated [α-32P]CTP-radiolabeled Pol IIo as well as a significant amount of Pol IIa (Figure 1A, lane 4). In the presence of α-amanitin, radiolabeled RNA was not detected on immunoprecipitated polymerase molecules. Thus, mAbs H5 and H14 can recognize a population of phosphorylated polymerase molecules that is transcriptionally engaged in the in situ nuclear run-on assay. Figure 1.(A) Transcriptionally active polymerase molecules are recognized by mAbs H5 and H14. 32P-labeled RNA was covalently cross-linked to transcriptionally active polymerase molecules in isolated nuclei (see Materials and methods). A nuclear extract prepared from these nuclei was used for immunoprecipitation with mAb H5 (specific for the hyperphosphorylated pol II) and mAb H14 (specific for multiple phosphorylated forms of pol II), mAb H22 (control IgM) and mAb M2 (control IgG). IIo, hyperphosphorylated largest subunit of RNA polymerase II; IIa, hypophosphorylated largest subunit of RNA polymerase II; IIc, second largest subunit of RNA polymerase II. Numbers at left margin indicate apparent molecular weights in kDa. (B and C) Nuclear localization of pol II. OV-MZ-15 cells were immunostained with mAb H5 (B) or mAb H14 (C). The broad staining of the non-nucleolar portion of the nucleus is referred to in this communication as a meshwork. Overlaying this meshwork were multiple discrete sites of preferred staining we have termed ‘dots’. Bar = 5 μm. Download figure Download PowerPoint Polymerase II localizes to many sites within the nucleoplasm Next, we wanted to know the nuclear localization of pol II that is actively engaged in transcription. mAb H5 and mAb H14 have been shown to stain mammalian cells broadly (Bregman et al., 1995). Figure 1B and C indicates a staining of human ovarian cancer cells (OV-MZ-15; Möbus et al., 1994) with antibodies mAb H5 (Figure 1B) and mAb H14 (Figure 1C). Broad staining of the entire non-nucleolar portion of the nucleus was observed, suggesting a wide distribution of pol II and, by inference, of transcription throughout the nucleoplasm. In this communication, we refer to this nucleoplasm-wide staining as a meshwork because at higher resolution of the image, staining was not uniformly diffuse, but instead appeared as a very dense fibrogranulous structure. Overlaying the meshwork were multiple discrete sites of preferred staining distinguishable from a classic speckle staining pattern. These discrete sites were very numerous in several tested cell lines, with an average number of 100–200 per cell in a given focal plane (suggesting up to 1000 per nucleus) and were observed throughout the entire nucleoplasm (with the exception of nucleoli). Therefore, preferred sites of staining for pol II were noticeably more numerous per nucleus than the number of speckles per nucleus. Most sites detected by pol II-specific antibodies were also smaller in appearance than classic speckles. To distinguish linguistically this latter pattern of pol II localization from that classically referred to as speckles, we have termed these multiple sites of pol II localization nuclear ‘dots’. Polymerase II localization pattern is distinct from speckles Speckles are frequently experimentally defined by staining with antibodies specific for splicing factors. One such factor, SC35, has been prominently localized to speckles (Fu and Maniatis, 1990; Spector et al., 1991). Figure 2A–C compares the staining pattern of cells stained with both pol II- and SC35-specific antibodies. Both antibodies stained broadly throughout the nucleoplasm in a meshwork pattern. Although the meshwork was much more visible with the pol II antibody in optical sections, the two patterns of staining overlapped extensively, suggesting that a portion of the SC35 appears to be localized in the vicinity of polymerase. Superimposed on this meshwork pattern were the prominently staining speckles of SC35 (Figure 2A, green) or the nuclear dots of pol II (Figure 2B, red). A few of the speckles overlapped with the ‘dots’ of polymerase II (overlay in Figure 2C); for the most part, however, the dots of preferred pol II localization and the SC35-defined speckles did not co-localize. Our result suggests that the bulk of polymerase II is not localized to speckles and raises questions about models that propose speckles as the sites of active transcription and RNA processing. Figure 2.Localization of pol II, BrU incorporation, and the splicing factor SC35. (A–C) Localization of pol II relative to the splicing factor SC35 by confocal microscopy. Immunofluorescence staining of SC35 (A, green) was compared with that of pol II as detected by the mAb H5 antibody (B, red; overlay in C). (D–L) Localization of nascent RNA relative to pol II by confocal microscopy. In situ RNA synthesis was localized by incorporation of BrUTP for 30 min and detection of incorporated BrU via the use of an antibody directed against BrU (D, G and J, green). Pol II was localized in the same cells by immunofluorescence staining with the mAb H5 (E, H and K, red). Co-localization of antibodies would appear yellow in overlaid panels (F, I and L). (D–F) Double localization in CaSki cells. Nuclei with appreciable BrUTP incorporation (top) and minimal BrUTP incorporation restricted to the nucleoli (bottom) are shown. (G–I) Double staining in a second human cell line OV-MZ-15. Note that in some double-stained foci of both cell lines, staining of pol II and BrU overlap but are not exactly the same size, shape, or intensity. (J–L) Arresting transcription causes a redistribution of polymerase II. Pol II-directed transcription was inhibited by treatment of CaSki cells with 1 mg/ml α-amanitin during an in situ transcription experiment. BrU incorporation was severely curtailed and bright staining was limited to nucleoli (J, green). Pol II staining was highly concentrated in speckle-like domains (K, red; overlay in L). Bar = 1 μm. Download figure Download PowerPoint Uridine incorporation and pol II overlap both in a meshwork throughout the nucleoplasm and in many preferred sites To confirm that the pattern of staining detected with pol II-specific antibodies represented sites of transcription, double-label experiments were performed in which the pattern of pol II staining using antibody mAb H5 was compared with sites of UTP incorporation during an in situ transcriptional run-on experiment (Figure 2D–I). Nascent transcripts were labeled with BrU and visually detected via the use of anti-BrU antibodies. Human CaSki (Figure 2D–F) and OV-MZ-15 (Figure 2G–I) cells were used for this experiment. For CaSki cells, two patterns of staining were observed that are represented by the two cells shown in Figure 2D–F. One pattern was seen in 60–80% of the cells and is represented by the top cell in Figure 2D. In these cells, nascent RNA as detected by confocal microscopy was localized throughout the nucleus in a pattern very similar to that of the pol II staining shown in Figure 2E (i.e. a nucleoplasm-wide meshwork overlaid with many sites of preferred staining). OV-MZ-15 cells demonstrated localization patterns of pol II and BrU incorporation similar to that of CaSki cells (Figure 2G–I). Both pol II and nucleotide incorporation occurred throughout the nucleoplasm in a complicated pattern. The merged views (Figure 2F and I) demonstrated considerable overlap of the two staining patterns (yellow) with the exclusion of nucleoli (marked as arrows). Because immunofluorescence can only provide an approximation of the actual extent of co-localization of two antigens with immunofluorescence patterns as complex as those in Figure 2D–I, a conservative interpretation of these patterns suggests that both pol II and transcription are broadly dispersed throughout the nucleoplasm in a particulate, non-speckled pattern. The underlying reason for both a meshwork and ‘dotted’ distribution of both pol II and BrUTP incorporation is unclear. Given the limits in resolution of light microscopy, the meshwork pattern could represent the transcription of single, moderately transcribed genes, whereas the dots could represent either clusters of genes or individual high-activity transcription units. Regardless of their origin, the similar appearance and location of both incorporation and polymerase suggests that both regions represent active sites of pol II-directed transcription and that both are clearly distinguishable from speckles. Close examination of multiple double-labeling experiments indicated that the discrete sites of pol II and BrU staining in cells demonstrating nuclei-wide BrU incorporation were not absolutely coincident. The size, shape or intensity in some of overlapping domains are not exactly the same. In particular, it was usually possible to observe a subfraction of pol II staining that had little associated BrU incorporation which presumably represented the subpopulation of pol II that was not actively engaged in the transcription (Kim et al., 1997). Moreover, the loci of nucleoplasmic BrU incorporation demonstrated considerable overlapping pol II staining. In addition to H5, mAb H14, which recognizes various phosphorylated forms of the pol II large subunit, was also used in co-localization of pol II and its transcripts. In both CaSki and OV-MZ-15 cells, H14 staining appeared identical to H5 pattern as a meshwork overlaid with discrete nuclear dots which overlapped with BrU staining, suggesting that different phosphorylated forms of pol II may exist in transcription domains. This result agrees with the results shown in Figure 1A that both antibodies can recognize transcriptionally engaged polymerase molecules. Pol II localizes to speckles in cells not actively involved in transcription A second pattern of in situ transcription was observed with CaSki, but not OV-MZ-15 cells, and is represented by the bottom cell in Figure 2D–F. This cell, representing 20–40% of the population, stained minimally with the anti-BrU antibody, indicating a poor incorporation of BrU. Incorporation in these cells was restricted to nucleoli when detected with phase-contrast microscopy (data not shown). In this second subpopulation of cells, pol II staining was concentrated into minimal domains that resembled speckles. A speckled distribution of pol II has been reported previously for MDCK cells (Bregman et al., 1995). In this latter study, speckles were preferentially seen in cells within the early G1 or late G2 phases of the cell cycle, in ∼20% of the cells within an unsynchronized cell population. The data in Figure 2D–F indicated that the pattern of pol II localization depended on the distribution of nascent transcripts, those cells that demonstrated active, nucleoplasm-wide transcription demonstrated nucleoplasm-wide pol II localization, suggesting that in these cells the staining pattern of pol II-specific antibodies represents sites of transcription of pol II transcribed genes. In contrast, the ‘speckle’ staining pattern for pol II was only observed in cells in which transcription was limited to the nucleoli, suggesting an alteration in localization of pol II, depending on the ability to detect numerous nascent transcripts. It should be noted that although mAb H5 recognizes hyperphosphorylated polymerase II, it has been shown to be capable of detecting pol II in cells arrested for transcription immediately before or after mitosis or with drug treatment (Bregman et al., 1995), indicating that the phosphorylated CTD epitope is still present under these conditions. In agreement with this interpretation, cells with and without appreciable BrU incorporation had approximately equal staining with mAb H5 (compare the two cells in Figure 2E), suggesting maintenance of the phosphorylated pol II epitope detected by this antibody in cells not actively incorporating BrU. We interpret the lack of nucleoplasm-wide pol II staining and BrU incorporation in the minor cell population to reflect minimal pol II-directed transcription in these cells. Alternative interpretations, including lack of permeability to both anti-pol II antibodies and nucleotide analogs, or inaccessibility of antigens to antibodies, seem unlikely because of the ability of pol II actively to decorate speckles in these cells as well as the ability to detect nucleotide incorporation into nucleoli in the same population. In vitro splicing has recently been shown to be inhibited if all uridines (U) within the utilized precursor RNA were replaced with BrU, presumably due to a strict requirement for U in consensus splicing sequences (Wansink et al., 1994). To address possible problems in factor localization arising from the use of a nucleotide analog, we compared the staining patterns of BrU, pol II and SC35 in CaSki and OV-MZ-15 cells during in situ transcription experiments using mixtures of UTP and BrUTP in various ratios, or in the total absence of BrUTP. The BrU staining pattern remained the same when the ratio of UTP:BrUTP was varied over a 4-fold range (data not shown), indicating minimal impact on the sites of transcription as BrUTP was added. More importantly, the pattern of pol II and SC35 staining remained the same when cells were transcribed in the absence or presence of BrUTP (data not shown). Thus, the distribution patterns of pol II and SC35 were not affected by the presence of BrU during an in situ transcription experiment. Inactivation of transcription localizes pol II to speckles The above experiments suggested a correlation between transcriptional state and the localization of polymerase II within the nucleoplasm. To better correlate the two, experiments were performed in which transcription was halted by either treatment with α-amanitin or heat shock (Figures 2J–L and 3, respectively). When α-amanitin was added to the in situ transcription of CaSki cells at a concentration sufficient to inhibit only polymerase II, BrU incorporation was severely curtailed in the entire population and bright staining was limited to nucleoli (Figure 2J). In all cells, pol II staining was highly concentrated in a minimal number of centers, to produce a pattern closely resembling that of speckles (Figure 2K and L). Thus, when pol II activity was inhibited and the synthesis of pre-mRNA arrested, pol II staining was highly localized. Figure 3.Heat shock causes a redistribution of polymerase II in MDCK cells. When cells were fixed and then permeabilized with non-ionic detergent, mAb H5 normally stained a meshwork, non-speckled pattern (A). Cells subjected to a heat shock of 45°C for 1 h, and stained with the same antibody showed a speckled pattern (B). After recovery at 37°C for 1 h, the speckled distribution of pol II disappeared and the meshwork staining pattern was restored (C); recovery at 0°C maintained pol II staining as a speckled pattern (D). Bar = 10 μm. Download figure Download PowerPoint Heat shock also inhibits pre-mRNA production. When MDCK cells were subjected to a heat shock of 45°C for 1 h, pol II distribution as monitored by staining with the mAb H5 antibody was concentrated into speckles as distinguished from the nucleoplasmic meshwork staining in cells at normal temperature (compare Figure 3B with Figure 3A). If permitted to recover at 37°C for 1 h, the speckled distribution of pol II disappeared and the normal meshwork staining pattern was restored. Recovery of pre-heat shock distribution of pol II required normal temperature; recovery at 0°C caused maintenance of pol II staining in a speckled pattern (Figure 3C and D, respectively). As with α-amanitin treatment, pol II distribution was throughout the nucleoplasm under conditions permitting transcription, but was more localized into a speckle-like pattern when pre-mRNA production was inhibited. The reversible recompartmentalization of pol II upon heat shock suggests a possible cycling of polymerase between speckles and more widely distributed sites of transcription. To confirm that the minimal staining domains of pol II detected when transcription was arrested actually were speckles, the staining pattern of pol II and SC35 were compared in CaSki cells under conditions of in situ transcription (Figure 4). As before, pol II staining was observed in different patterns in subpopulations of cells: a broad meshwork was observed in a majority of cells with pronounced ‘speckles’ in a minority of cells (speckles marked with arrows in Figure 4A). In addition, there were cells in the population that demonstrated an intermediate pattern of pol II distribution. The speckles of pol II correlated well with the speckles of SC35 (Figure 4B). Interestingly, SC35-detected speckles were most pronounced in those cells demonstrating pol II speckles. In those cells in which pol II was more dispersed to the meshwork and nuclear dots, the SC35 speckles were less pronounced, suggesting that SC35, like pol II, may redistribute dynamically within nuclei depending on transcription (see also Figure 6). Figure 4.Dynamic localization of pol II and the splicing factor" @default.
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• W2066016774 title "Dynamic relocation of transcription and splicing factors dependent upon transcriptional activity" @default.
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• W2066016774 hasConceptScore W2066016774C70721500 @default.
• W2066016774 hasConceptScore W2066016774C78458016 @default.
• W2066016774 hasConceptScore W2066016774C86339819 @default.
• W2066016774 hasConceptScore W2066016774C86803240 @default.
• W2066016774 hasConceptScore W2066016774C95444343 @default.
• W2066016774 hasIssue "6" @default.
• W2066016774 hasLocation W20660167741 @default.

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