Article |
Address correspondence to Dr. Veena K. Parnaik, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad-500 007, India. Tel.: 91-40-27192614. Fax: 91-40-27160591/27160311. E-mail: veenap{at}gene.ccmbindia.org
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Abstract |
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Key Words: nuclear lamina; lamin A; splicing factor compartments; RNA polymerase II transcription; nuclear organization
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Introduction |
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Lamins belong to the intermediate filament family of proteins and contain a characteristic central -helical rod domain, flanked by relatively flexible NH2- and COOH-terminal segments. All three domains are essential for lamin assembly and functions (for review see Stuurman et al., 1998). Deletion of the NH2-terminal domain of human lamin A (Spann et al., 1997; Moir et al., 2000a) or Xenopus lamin B1 (Ellis et al., 1997) leads to defects in lamina assembly, disruption of the lamina, and inhibition of DNA replication. Mutations in human lamin A cause debilitating diseases such as Emery-Dreifuss muscular dystrophy, cardiomyopathy, partial lipodystrophy and axonal neuropathy (Bonne et al., 1999; Fatkin et al., 1999; Cao and Hegele, 2000; Shackleton et al., 2000; De Sandre-Giovannoli et al., 2002). A few of these lamin mutant proteins cause gross defects in the peripheral lamina and also assemble aberrantly, but other mutants do not show an obvious phenotype (Östlund et al., 2001; Raharjo et al., 2001; Vigouroux et al., 2001).
The presence of morphologically distinct nuclear compartments that are enriched for specific proteins is now well established (for reviews see Spector, 1993; Lamond and Earnshaw, 1998). RNA splicing factors are present in high concentrations in compartments or speckles called splicing factor compartments (SFCs)* that correspond at the electron microscopic level to interchromatin granule clusters (IGCs) and are also dispersed in the nucleoplasm on perichromatin fibrils (PFs), which contain nascent transcripts (for reviews see Fakan and Puvion, 1980; Spector, 1993; Fakan, 1994). The splicing of pre-mRNAs occurs concomitantly with transcription on PFs (Beyer et al., 1988) and away from, or at the periphery of, SFCs for most transcripts (Jackson et al., 1993; Wansink et al., 1993; Cmarko et al., 1999). Transcription by RNA polymerase II (pol II) has been visualized on hundreds of small foci throughout the nucleoplasm (Jackson et al., 1993; Wansink et al., 1993; Bregman et al., 1995). The SFCs are dynamic compartments involved in the storage/recruitment of splicing factors (Misteli et al., 1997). Their size can change depending on RNA splicing or transcription levels in the cell; for example, they become considerably enlarged due to reduced dissociation of splicing factors in the presence of transcriptional inhibitors (Carmo-Fonseca et al., 1992; Spector, 1993), in pathological conditions (Fakan and Puvion, 1980), or upon inhibition of splicing (O'Keefe et al., 1994).
The gene-specific positioning of transcription sites with respect to SFCs (Smith et al., 1999) and recruitment of splicing factors from SFCs upon gene activation (Misteli et al., 1997) point to significant spatial coordination of transcription and pre-mRNA splicing. A key issue that has not yet been resolved is the importance of nuclear architecture in the spatial organization of transcription and pre-mRNA splicing. It has been proposed that SFCs are generated by interactions with the nucleoskeletal framework (Kruhlak et al., 2000), or, alternatively, that self-organization of splicing factors leads to the assembly of SFCs (Misteli, 2001). The association of transcription sites or active pol II with an insoluble nuclear framework or matrix has been well documented (Jackson et al., 1993; Wansink et al., 1993; Kimura et al., 1999; Wei et al., 1999), several transcription factors have been localized to the nuclear matrix (for review see Stein et al., 2000), and SFCs have also been observed to be attached to a detergent-insoluble nuclear structure (Spector, 1993). Importantly, Hendzel et al. (1999) have demonstrated the presence of an underlying protein architecture in IGCs that physically connects the relatively dispersed granules within the cluster, by using energy transmission electron microscopy in intact cells and thus avoiding the problems associated with typical nuclear matrix isolation protocols (Pederson, 2000; Nickerson, 2001). The main candidate protein constituents of the nuclear framework are the lamins, previously identified at the nuclear periphery but now also observed in the nuclear interior, and actin, which can bind to snRNPs (Nakayasu and Ueda, 1984). A role for lamins in controlling gene expression has been proposed earlier (Wilson, 2000), and in vitro binding of lamin A to the retinoblastoma protein, an important transcriptional regulator, has been reported (Ozaki et al., 1994). More recently, an NH2-terminal deletion lamin A mutant, NLA, that disrupts the lamina has been observed to inhibit transcription (Spann et al., 2002). However, the involvement of internal lamins in the spatial organization of transcription or splicing has not been demonstrated.
The aim of this study is to understand the functional role of internal lamin A/C speckles that have been observed to colocalize with SFCs in a variety of cell types using a monoclonal antibody to rat lamin A that has certain unique properties (Jagatheesan et al., 1999). This antibody, mAb LA-2H10, exclusively stains intranuclear speckles in interphase cells without labeling the peripheral lamina, and specifically recognizes only lamins A and C in immunoblots of cellular fractions (the epitope region spanning amino acids 171246, which has no significant homology with nonlamin proteins, is common to both lamins). mAb LA-2H10 does not cross-react with any other proteins from whole cell lysates in the molecular mass range of 14250 kD (unpublished data). As the exclusive reactivity of mAb LA-2H10 toward speckles is retained when cells are detergent and nuclease treated to reveal the nucleoskeletal framework, we have attributed this reactivity to the differences in lamin protofilament interactions at the periphery and at internal sites. We have recently demonstrated that muscle differentiation is accompanied by rearrangements in the organization of lamin A speckles, which appear to be controlled by cell cycleregulated pathways (Muralikrishna et al., 2001). In the present study, we have investigated the possibility of a direct correlation between the organization of lamin A/C speckles, SFCs, and transcriptional events by first examining the effects of inhibitors of pol II transcription. Our results show that lamin A/C speckles reorganize to form enlarged foci in the presence of transcriptional inhibitors, as do the RNA splicing factors SC-35 and U5-116 kD. Removal of a reversible inhibitor results in rapid, synchronous redistribution of lamin A/C speckles and splicing factors with reactivation of transcription. Second, we have observed that expression of NH2-terminally tagged lamin A (His or FLAG) or lamin C (His), but not FLAGlamin B1, leads to the disruption of lamin A/C speckles and SFCs, accompanied by a reduction of active pol II and BrUTP incorporation into nascent RNA, without discernible effects on the incorporation of tagged lamin A or C into the nuclear periphery or disruption of the endogenous lamina at the nuclear rim. These results suggest a close association between the internal lamin A/C network and the organization of splicing factors and transcriptional events.
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Results |
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We have earlier demonstrated the presence of lamin A/C speckles that are labeled with the antibody mAb LA-2H10 and colocalize with RNA splicing factors such as SC-35 and U5-116 kD in several mammalian cell types (Jagatheesan et al., 1999). SC-35 is an essential non-snRNP splicing factor (Fu and Maniatis, 1990), whereas U5-116 kD is a 116-kD protein that is a component of U5 snRNPs (Fabrizio et al., 1997). Both proteins are concentrated in speckles and also exhibit diffuse nucleoplasmic localization, but to different levels. To explore the possibility that lamin A/C speckles might be modified by transcriptional inhibitors, we have examined the effects of -amanitin and DRB on the distribution of lamins. When HeLa cells were incubated with
-amanitin and then stained with mAb LA-2H10, a striking redistribution of lamin speckles into enlarged foci was observed (Fig. 1), similar to that seen with SC-35 and U5-116 kD. There were, however, no changes in the typical peripheral pattern of A-type lamins stained with LA-2B3. To confirm the efficacy of
-amanitin treatment, the levels of pol II activity were detected by staining with mAb H5. This antibody recognizes only the active, hyperphosphorylated form of pol II, termed pol IIO, and stains 7080% of a population of dividing cells in a diffuse pattern of tiny foci that correspond to centers of active transcription, and
2030% of cells in a speckled pattern, which is more pronounced in cells prepermeabilized with 1.5% Triton X-100 (Bregman et al., 1995). However, as prepermeabilization weakened the diffuse signal, our samples were fixed under standard conditions. Labeling with mAb H5 was reduced to low levels in cells treated with
-amanitin. Furthermore, the incorporation of BrUTP into newly synthesized RNA transcripts by pol II was abolished in the nucleoplasm of permeabilized cells that had been preincubated with
-amanitin before the addition of ribonucleotides, whereas pol Igenerated transcripts continued to be detected in nucleoli, as documented earlier (Wansink et al., 1993). Because lamins A/C bind to the inner nuclear membrane protein emerin (Clements et al., 2000), the effects of
-amanitin on emerin staining were examined. Both treated and untreated cells showed a uniform nuclear rim labeling for emerin. There were also no detectable effects on the distribution of nuclear pore complexes (NPCs), as observed by staining with mAb 414, which labels a major group of O-glycosylated NPC proteins (Davis and Blobel, 1986). Similar effects of
-amanitin on lamin speckles were also observed with BHK-21 cells (unpublished data).
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Absence of lamin speckles in cells expressing modified lamin A/C
To further understand the functions of lamin A/C speckles, we wished to examine the effects of disrupting the internal lamina organization, without causing gross abnormalities in the peripheral lamina or nuclear morphology, by overexpressing suitably modified lamin A proteins in cultured cells. Both the NH2-terminal domain and COOH-terminal CaaX motif of lamin A have been shown to be essential for lamin assembly and functions and necessary to direct exogenously expressed lamin A to the peripheral lamina (Spann et al., 1997; Izumi et al., 2000; Moir et al., 2000a). In exploratory experiments with NH2-terminally tagged mutants deleted in a putative chromatin-binding domain encompassing amino acid residues 396401 (Taniura et al., 1995), we observed a considerable loss of lamin A/C speckles in transfected cells. Surprisingly, similar observations were made with the wild-type NH2-terminally tagged constructs. Hence, we have investigated the properties of wild-type tagged lamin A constructs in detail.
The constructs that have been analyzed in this study are as follows. Hislamin A and Hislamin C have an additional six histidine residues at the NH2 terminus of full-length lamin A or C respectively, whereas FLAGlamin A has the FLAG epitope in a similar position in lamin A (sequences are given in the Materials and methods). The recombinant proteins have been visualized in transfected cells by using antibodies that detect 6X His or FLAG sequences. The specificity of these antibodies was confirmed in immunoblots of HeLa cell lysates, as shown in Fig. 3 A. The tagged proteins were labeled by their respective antibodies in transfected cell lysates, and there was no cross-reactivity with untransfected cell lysates. Both prelamin A and mature lamin A could be detected in lysates of tagged lamin Atransfected cells. Analysis with mAbs LA-2H10 and LA-2B3 did not indicate any obvious changes in the mobilities of lamins in transfected cells as compared with untransfected cells. As the efficiency of transfection was 20%, a significant increase in the abundance of lamins was not detected in these blots. As shown in Fig. 3 B, exogenous lamins were incorporated into the nuclear periphery to give a smooth rim staining in cells transfected with Hislamin A or FLAGlamin A. Hislamin C occasionally formed small internal aggregates or round patches toward the periphery in addition to a rim; the internal aggregates were more prominent in samples examined 1012 h after transfection (unpublished data), consistent with earlier reports on the assembly properties of exogenous lamin C (Goldman et al., 1992; Bridger et al., 1993; Pugh et al., 1997).
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Disruption of SFCs and pol II transcription upon overexpression of modified lamins A/C
The disappearance of lamin A/C speckles in cells expressing NH2-terminally tagged lamin A or C prompted us to investigate the organization of SFCs in these cells. When HeLa cells were transfected with Hislamin A or C and labeled with antibody to SC-35, 60% of transfected cells showed a reduction of SC-35 staining, as illustrated in Fig. 6 A. Exogenous expression of Hislamin A or C also caused a loss of U5-116 kD in 60% of transfected cells. The depletion of splicing factors upon the disruption of lamins is unexpected and suggests that lamin speckles are important for the maintenance of SFCs. Splicing factors are also reduced in
NLA-injected cells (Spann et al., 2002); on the other hand, other treatments such as overexpression of Clk/STY protein kinase result in the redistribution of splicing factors from speckles to a diffuse pattern (Colwill et al., 1996; Sacco-Bubulya and Spector, 2002). Cells expressing tagged lamins did not exhibit any significant changes in the distribution or morphology of Cajal bodies, visualized by p80 coilin staining, which is consistent with the absence of association of lamin A/C speckles with these compartments and rules out a generalized collapse of intranuclear compartments. The expression of FLAGlamin A gave results similar to those obtained with Hislamin A; on the other hand, splicing factor distribution was not altered in cells expressing FLAGlamin B1 (unpublished data).
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Discussion |
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Dynamic association of lamin A/C and SFCs
SFCs are dynamic compartments involved in the storage and recruitment of splicing factors. Splicing factor assemblies containing SF2/alternate splicing factor (ASF) are recruited from SFCs to transcription sites in vivo upon gene activation (Misteli et al., 1997). However, when transcription is inhibited there is a decrease in speckle dynamics, and budding and transport of smaller domains derived from speckles are no longer seen. There is a concomitant reorganization of speckles to larger and more spherical foci. Removal of the reversible transcriptional inhibitor DRB causes restoration of transcription by pol II, and, simultaneously, splicing factors such as SC-35 redistribute within the nucleus to their normal organization (Bregman et al., 1995). We have observed a coordinate redistribution of lamin A/C and SC-35 upon reactivation of transcription beginning within 10 min of removal of DRB. Thus lamins, in association with SFCs, can move relatively rapidly in response to signals that also cause the relocalization of splicing factors to sites of transcription, and might be regulated by similar signaling events. The dynamic reorganization of lamins A/C in response to transcriptional inhibition is consistent with a dynamic remodeling of potential structural components of the nucleoplasm in response to changing nucleoplasmic transcription.
We speculate that the redistribution of lamins may involve the depolymerization and repolymerization of lamin filaments. We have earlier observed that the speckles are resistant to detergent and/or salt extraction and nuclease digestion (Jagatheesan et al., 1999; Muralikrishna et al., 2001); hence speckles are likely to be composed of lamin filaments, though these may be in a different configuration than those found at the nuclear periphery. The head-to-tail arrangement of lamin dimers to form a protofilament has been well established; however, very little is known about the possible structures that can be adopted during lateral assembly of protofilaments to form the mature filament (Stuurman et al., 1998). The comparatively rapid redistribution of lamin A speckles observed in this study suggests that this pool of lamin A is in dynamic equilibrium, unlike the relatively static lamin framework at the nuclear rim, which dissociates only during mitosis. Further examples of lamin redistribution include the reorganization of lamin speckles to a uniform network during muscle differentiation (Muralikrishna et al., 2001); these rearrangements appear to occur specifically in muscle cells, probably due to the unique morphology of the multinucleated myofiber that is formed. The association of lamins with DNA replication centers in S phase cells is an example of cell cycledependent changes in lamina organization (Moir et al., 1994). In live cell studies on lamin dynamics, GFPlamin A does not form speckles, possibly due to the large size of the GFP tag, and is primarily incorporated into the nuclear periphery (Broers et al., 1999; Moir et al., 2000b). As lamin speckles are highly insoluble structures, they do not copurify with IGCs in stoichiometric amounts, though small quantities of lamins have been detected in purified IGCs (Mintz et al., 1999).
Implications for the spatial organization of RNA splicing and transcription
A close coordination between the processes of RNA splicing and transcription is indicated by several lines of evidence, such as the cotranscriptional splicing of nascent transcripts (Beyer et al., 1988) and the association of the COOH-terminal domain of pol lI with a number of multiprotein complexes involved in transcription and pre-mRNA splicing (for review see Hirose and Manley, 2000). Furthermore, studies with inhibitors of splicing or transcription indicate that blocking one process generally affects the other (Carmo-Fonseca et al., 1992; Spector, 1993; O'Keefe et al., 1994). The movement of ASF-enriched domains from SFCs toward specific transcription sites upon BK viral gene activation (Misteli et al., 1997) is suggestive of a directed targeting of the splicing machinery to required locations. The detailed analysis of the mobility of GFP-tagged nuclear proteins within the nucleoplasm of live cells by fluorescence recovery after photobleaching and fluorescence loss in photobleaching techniques indicates substantial mobility of these proteins in the nucleus (Kruhlak et al., 2000; Phair and Misteli, 2000). However, this mobility is considerably less than that of GFP alone, and Kruhlak et al. (2000) suggest that ASF-containing structures behave as though they are physically impeded by frequent and transient associations with nucleoskeletal elements. This is further substantiated by single particle tracking studies with the splicing factor U1 snRNP whose dynamics within SFCs is much slower than in aqueous solution, suggesting transient binding to immobile sites (Kues et al., 2001).
Our observations on the synchronous, reversible changes in lamin speckles and SFCs due to the inhibition of transcription and, conversely, the down-regulation of transcription after disruption of lamin speckles provide compelling evidence for a role for lamin speckles in the organization of transcription and splicing. We propose that lamin A/C speckles are part of a dynamic structure that can be rapidly modulated by specific signaling events to spatially organize mRNA splicing and pol II transcription. Protein kinase activities have been proposed to functionally link the transcription and splicing machineries, primarily by the phosphorylation of arg-serrich splicing factors that assist in their recruitment to splicing sites (Misteli et al., 1998). Though lamin A does not have a distinct arg-ser domain, phosphorylation might control lamin localization, albeit indirectly, through interactions with other proteins; for instance, CrkRS has been shown to be tightly bound to the nuclear matrix and can also phosphorylate the COOH-terminal domain of pol II (Ko et al., 2001). Other structural proteins may perform similar functions to those proposed for lamins A/C; for example, lamins B1/B2 might compensate for the lack of A-type lamins in undifferentiated cells or under pathological conditions, or actin might also play a structural role in the nucleus. The association of factors with a structural framework would undoubtedly allow for greater coordination of regulatory events in the nucleus as well as higher specificity and efficiency.
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Materials and methods |
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Cell culture and DNA transfection
HeLa cells were grown on coverslips to 70% confluency in Petri dishes containing DME supplemented with 10% FCS. DNA transfections were performed with lipofectamine (Invitrogen) according to the manufacturer's instructions. Cells were processed for immunofluorescence microscopy 24 h after transfection. Transfection efficiencies were 20%. Cells were treated with the transcriptional inhibitor
-amanitin at 50 µg/ml for 5 h and DRB at 25 µg/ml for 3 h.
Antibodies
Antibodies to recombinant rat lamins used in this study and characterized in detail previously are mAb LA-2H10, which recognizes intranuclear lamin A speckles, and mAb LA-2B3, which stains the nuclear periphery (Jagatheesan et al., 1999). SC-35 mAb (Fu and Maniatis, 1990) was provided by J. Gall (Carnegie Institution of Washington, Baltimore, USA); rabbit polyclonal antibodies to U5-116 kD (Fabrizio et al., 1997) were from R. Lührmann (University of Marburg, Marburg, Germany); and p80 coilin (Bohmann et al., 1995) was from A. Lamond (University of Dundee, Dundee, Scotland). A rabbit polyclonal antibody to recombinant TBP was provided by P. Bhargava (Centre for Cellular and Molecular Biology). His and FLAG mAbs were from CLONTECH Laboratories, Inc. and Sigma-Aldrich, respectively; rabbit polyclonal antibodies to His and pol IIi were from Santa Cruz Biotechnology Inc.; and mAb 414 to NPC proteins and mAb H5 to pol IIO were from Berkeley Antibody Co. An emerin mAb was obtained from Novocastra Laboratories Ltd. and a rat mAb to BrdU was from Sera Laboratories.
Immunofluorescence microscopy
HeLa cells were washed with PBS and then fixed by treatment with 3.5% formaldehyde for 15 min followed by 0.5% (vol/vol) Triton X-100 for 6 min at room temperature, or with methanol at -20°C for 10 min, as indicated. Cells were then incubated with 0.5% gelatin in PBS for 1 h followed by incubation with first antibody for 1 h and then FITC-conjugated second antibody for 1 h at room temperature for single labeling experiments. For double labeling experiments, fixed cells were incubated with the first primary antibody followed by biotinylated second antibody and avidin-Cy3, and then with the next primary antibody and species/subtype-specific FITC-conjugated second antibody. For double labeling experiments with antibodies of the same subtype, one of the primary antibodies was directly biotinylated for detection with Cy3. For triple labeling studies, fixed cells were either stained sequentially with anti-His antibody and antimouse AMCA conjugate, followed by mAb LA-2H10 and Texas redconjugated antimouse IgM, and then biotinylated SC-35 antibody and avidin-FITC; or with anti-His antibody and FITC-conjugated antimouse IgG, followed by mAb H5 and Texas redconjugated antimouse IgM, and then biotinylated LA-2H10 and Alexa®-350. Samples were mounted in Vectashield (Vector Laboratories) containing 1 µg/ml DAPI. There was no cross-reactivity of the fluorescent second antibodies in control experiments in which either primary antibody was omitted. Antibody conjugates were from Jackson ImmunoResearch Laboratories, Molecular Probes, or Vector Laboratories. Confocal laser-scanning immunofluorescence microscopy was performed on a Meridian Ultima scan head attached to an Olympus IMT-2 inverted microscope fitted with a 100X, 1.3 NA or 60X, 1.4 NA objective lens, with excitation at 515, 488, and 351364 nm (Argon ion laser). Image analysis, including crossover subtraction, two-dimensional quantitation of pol IIO fluorescence, and estimation of colocalized speckles, was done using DASY master program V4.19 (Meridian Instruments Inc.), and images were assembled using Adobe Photoshop 5.0®. Quantitative analysis was performed by inspection of n = 100 cells per sample. To reduce photobleaching of UV-sensitive dye conjugates, triple-labeled samples were viewed on an Olympus BX60 fluorescence microscope with a cooled camera device, and images were analyzed using ImagePro software.
RNA synthesis
The incorporation of BrUTP into nascent RNA was measured by a standard protocol (Wansink et al., 1993). HeLa cells (untransfected or 24 h after transfection) were rinsed once with PBS and once with a glycerol-containing buffer (20 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 25% glycerol, 0.5 mM PMSF, and 0.5 mM EGTA). Cells were then permeabilized in glycerol buffer containing 0.1% Triton X-100 at room temperature for 3 min and washed once with glycerol buffer. Subsequently, cells were incubated in transcription mix (100 mM KCl, 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 0.5 mM EGTA, 25% glycerol, 1 mM PMSF, 2 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 0.2 mM BrUTP, and 25 U/ml RNAsin) for 4.5 min at 37°C. At the end of the transcription reaction, cells were rinsed twice with cold PBS and then fixed in 3.5% formaldehyde in PBS. Fixed cells were labeled with anti-BrdU antibody as described above.
Extraction of nuclei
Cells were extracted by the relatively mild protocol described by De Conto et al. (2000), as this was found to be more suitable for cells after transfection. Transfected cells plated on coverslips were rinsed twice with TM buffer (50 mM Tris-HCl, pH 7.5, 3 mM MgCl2) and then incubated for 10 min on ice in TM buffer containing 0.4% Triton X-100, 0.5 mM CuCl2, and 0.2 mM PMSF. Cells were rinsed and incubated with DNase I (20 U/ml) and RNase A (20 µg/ml) for 20 min at 37°C in TM buffer. The samples were then treated with 2 M NaCl for 5 min on ice, washed with TM buffer, fixed with formaldehyde, and stained as described above.
Immunoblot analysis
HeLa cell lysates were analyzed by immunoblotting as described previously (Jagatheesan et al., 1999).
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Footnotes |
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* Abbreviations used in this paper: ASF, alternate splicing factor; DRB, 5,6-dichlorobenzimidazole riboside; IGC, interchromatin granule cluster; NPC, nuclear pore complex; PF, perichromatin fibril; pol II, RNA polymerase II; SFC, splicing factor compartment; TBP, TATA binding protein.
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Acknowledgments |
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Submitted: 26 April 2002
Revised: 25 October 2002
Accepted: 1 November 2002
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References |
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