Article |
Address correspondence to David L. Spector, Cold Spring Harbor Laboratory, 1 Bungtown Rd., Cold Spring Harbor, NY 11724. Tel.: (516) 367-8456. Fax: (516) 367-8876. E-mail: spector{at}cshl.org
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Abstract |
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Key Words: interchromatin granule cluster; transcription; pre-mRNA splicing; Clk/STY; nucleus
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Introduction |
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Although splicing factors localize to IGCs, pre-mRNA synthesis does not occur within these structures, but at PFs on the IGC periphery or at some distance away from the IGCs (Fakan, 1994; Cmarko et al., 1999). One recent report indicated overlap between sites of bromo-uridine incorporation and nuclear speckles (Wei et al., 1999); however, a significant proportion of the overlap likely corresponds to PFs (transcription sites) on the periphery of speckles. The majority of nucleotide incorporation and immunocytochemistry studies have shown that IGCs are not likely to be sites of transcription because they do not contain DNA, and they are not labeled by 3H-uridine incorporation (Turner and Franchi, 1987; Spector, 1990; Fakan, 1994). Cmarko et al. (1999) performed extensive analysis of bromo-UTP incorporation at the TEM level and did not detect transcription in IGCs. Furthermore, inhibition of RNA polymerase II transcription with -amanitin causes splicing factor recruitment to cease, and speckles become larger and more rounded (Carmo-Fonseca et al., 1992; Spector et al., 1993; Misteli et al., 1997). Therefore, enrichment of splicing factors in IGCs may be due to the fact that they are sites of complex formation and/or modification of splicing factors, or sites of splicing factor storage (Huang et al., 1994; Spector et al., 1993). In support of these possibilities, experiments in living cells revealed that hyperphosphorylation of splicing factor SF2/ASF on its RS-rich domain releases it from the IGCs for recruitment to active genes (Misteli et al., 1997). However, despite this advance, neither the structural organization nor the precise biological function of the IGCs is known.
Several SR protein kinases, including cdc2-like kinase (CLK)/STY 1, 2, 3, and 4 (Ben-David et al., 1991; Hanes et al., 1994; Howell et al., 1991) and SR protein kinases 1 and 2 (SRPK1 and SRPK2) (Gui et al., 1994; Wang et al., 1998; Yeakley et al., 1999), specifically phosphorylate RS domains. Clk/STY was isolated independently by several groups (Ben-David et al., 1991; Howell et al., 1991; Johnson and Smith, 1991) and was subsequently characterized as a LAMMER family kinase. LAMMER kinases include dual specificity kinases that can phosphorylate on tyrosine in addition to serine/threonine residues (Lindberg et al., 1992; Lee et al., 1996). When Clk/STY is overexpressed in cultured cells, SR proteins become hyperphosphorylated and the typical speckled immunolocalization of splicing factors is reorganized into a diffuse nucleoplasmic localization (Colwill et al., 1996b). In addition, Clk/STY has been shown to directly affect the activity of SR proteins; both hyper- and hypophosphorylation of SR proteins affect in vitro splicing activity (Prasad et al., 1999).
It is presently unclear why mammalian cells contain IGCs and whether their position in the nucleus reflects a spatial positioning that is essential for function. Time-lapse observations of nuclear speckles in living cells have shown that the position of IGCs is maintained over many hours (Misteli et al., 1997; Kruhlak et al., 2000), suggesting that they reside in predetermined locations. Such positioning may be a result of granules clustering upon a specific structural framework or around specific chromosomal regions. To investigate this possibility, we used overexpression of murine Clk/STY 1 as a method to completely disassemble IGCs in vivo. Ultrastructural analysis of such cells indicates that SR proteins are redistributed throughout the nucleus in small clusters, and no specific underlying structural IGC scaffold was revealed. Nascent transcripts are produced in cells without IGCs, but accumulation of splicing factors originating from an entirely nucleoplasmic pool onto pre-mRNA is significantly reduced and spliced mRNA is markedly reduced to undetectable.
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Results |
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Immunofluorescence localization with nonphosphoepitope antibodies against B" (Fig. 9, AC), SF2/ASF (Fig. 9, DI), and m3G (unpublished data) verified that these splicing factors precisely colocalized with GFP-Clk/STY(K190R) in the foci. We interpret these foci as regions of the speckles in which splicing factors accumulate because they are in a state of reduced phosphorylation and therefore cannot be released from the speckles. To confirm this hypothesis, we transfected A-431 cells with GFP-Clk/STY(K190R) and performed immunofluorescence using anti-SC35 antibody, which recognizes a phosphoepitope on SC35, and mAb104, which recognizes phosphoepitopes on a family of SR proteins (Roth et al., 1990). There was a dramatic reduction of immunolabeling with these phosphoepitope antibodies in the focal accumulations of GFP-Clk/STY(K190R), as noted by complete absence of labeling in these regions with mAb104 (Fig. 10, AC) and with anti-SC35 (Fig. 10, DI). This result confirms that unphosphorylated or hypophosphorylated proteins are present in the foci and might be unable to leave the speckles due to a lack of or reduced levels of phosphorylation.
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Discussion |
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Although it does not appear that IGC components, such as lamin A or actin, form filaments that provide a scaffold for clustering of interchromatin granules, it is possible that individual interchromatin granules may require these structural proteins as monomers or very short multimers in order to assemble a large number of protein and RNA components into particles. Alternatively, G-actin or lamin A monomers may be recruited to transcription sites as members of interchromatin granules. Once at transcription sites, they may multimerize into short filaments that may act as a scaffold for the assembly/disassembly of the transcription RNA processing complex. In support of this possibility, ß-actin and actin-related proteins are components of the mammalian SWI/SNF-like BAF (Brg-associated factor) complex, and binding of the BAF complex to the nuclear matrix in vitro is enhanced by phosphatidylinositol (4,5)-bisphosphate (PtdIns[4,5]P2), a lipid that regulates actin-binding proteins (Zhao et al., 1988). Furthermore, PtdIns(4,5)P2 and multiple phosphatidylinositol phosphate kinase (PIPK) isoforms have been localized to nuclear speckles in vivo by antibody labeling (Boronenkov et al., 1998). Recently, Percipalle et al. (2001) have shown that actin becomes associated with a Balbiani ring mRNA via a heterogeneous nuclear ribonucleoprotein (hrp36) at the site of transcription. Future studies will directly address the organization of individual interchromatin granules and the possible role of structural proteins in their assembly/disassembly.
We examined the effect of Clk/STY hyperphosphorylation on the release of a large number of protein constituents of IGCs, including many that do not contain the RS domain essential for phosphorylation by Clk/STY. Our finding that all proteins redistributed, regardless of the presence of an RS domain, is consistent with the proposal that transcription and RNA-processing factors may exist in the nucleus in a unitary particle called a transcriptosome (Gall et al., 1999), or alternatively, in multiple smaller complexes. However, it is currently unclear if such a particle is held together simply by proteinprotein interactions or in concert with other potential mechanisms. In this regard, it is particularly intriguing that upon overexpression of Clk/STY, the stable population of poly(A)+ RNA that is present in IGCs (Huang et al., 1994) becomes diffusely distributed throughout the nucleoplasm, whereas nascent transcripts at specific transcription sites do not redistribute. This finding raises the possibility that the stable population of poly(A)+ RNA that is localized to IGCs may have a role in maintaining the organization of pre-mRNAprocessing factors at these nuclear domains. These stable RNAs may represent the core organizing unit of individual interchromatin granules and the binding site for RNA-processing proteins. Studies are currently underway to purify and characterize these RNA molecules.
The release of splicing factors from IGCs by hyperphosphorylation makes these factors available for recruitment to sites of transcription and splicing (Misteli et al., 1997). Since not all interchromatin granules dissociate at once, regulatory mechanisms must influence the steady-state level of interchromatin granules within these structures as well as the rate of release of complexes into the nucleoplasmic pool. In this study, we tested whether an entirely nucleoplasmic pool of splicing factors, that presumably would be fully accessible to transcription sites, was sufficient for both recruitment and function. Interestingly, we found that disruption of IGCs did not affect synthesis of pre-mRNA on either a global or a specific level. However, IGC disassembly largely prevented accumulation of splicing factors on nascent transcripts at the site of transcription, and in doing so significantly reduced or abolished pre-mRNA splicing. Interestingly, in a previous study we have shown that microinjection of antisense oligonucleotides or antibodies to pre-mRNA splicing factors resulted in the rounding up of nuclear speckles and an inhibition of both transcription and pre-mRNA splicing (O'Keefe et al., 1994). In this study we show that this coordination can be disrupted by the break-up of nuclear speckles, suggesting that this nuclear structure plays some role in coupling these two processes.
We cannot completely exclude the possibility that reduction in splicing in vivo could be due to inactivation of splicing factors via their hyperphosphorylation rather than the loss of IGCs. For example, a recent in vitro study showed that Clk/STY directly affects the activity of SR proteins, and altering the phosphorylation state of these proteins either by hyper- or hypophosphorylation resulted in inhibition of splicing activity (Prasad et al., 1999). Overexpression of Clk/STY in vivo has also been shown to affect splice site selection on a reporter transcript (Duncan et al., 1997), although correlation with the extent of IGC disassembly was not reported. However, our data demonstrate that upon hyperphosphorylation of SR proteins, all components tested, including those that do not contain RS domains, were redistributed. Both an SR protein (SC35, Fig. 6 F) and a non-SR protein U2-B" (unpublished data) failed to accumulate at transcription sites, and there was a marked reduction in spliced product. It is therefore likely that in vivo the organization of IGCs is fundamentally linked to the phosphorylation state of SR proteins and hence to the ability of the processing machinery to perform pre-mRNA splicing. This possibility is further supported by previous studies showing that phosphorylation of SR proteins was linked to their release from IGCs and subsequent recruitment to transcription sites (Misteli et al., 1997). While pre-mRNA splicing can occur in vitro in the absence of intact IGCs, it is conceivable that nuclear extracts used for such experiments contain individual interchromatin granules that may be altered upon SR protein hyper- or hypophosphorylation, leading to decreased splicing activity.
The present study implicates IGCs in the assembly/maturation of the RNA-processing machinery into splicing-competent complexes or particles that must be maintained during transit to active genes for efficient targeting and function. Perhaps certain components of these complexes are responsible for recognizing newly synthesized pre-mRNA and/or stabilizing the association of the splicing machinery on pre-mRNA. Our finding that GFP-Clk/STY was recruited to transcription sites is consistent with the possibility that, in addition to regulating release of splicing factors from IGCs, it might also regulate/remodel splicing factor interactions during alternative or constitutive splicing.
Assuming that Clk/STY is responsible for phosphorylation events that lead to release of splicing factors from nuclear speckles, then a mutant kinase that lacks SR protein kinase activity would be expected to inhibit the release of splicing factors in vivo. As predicted, overexpression of GFP-Clk/STY(K190R) causes peripheral regions of nuclear speckles to become immobilized. Splicing factors accumulate in foci, and this ultimately leads to a reduction in splicing activity. Perhaps the mutant kinase is interacting with its substrates, but because it is unable to phosphorylate them, the result is sequestration of hypophosphorylated splicing factor complexes in foci. Examination of these regions in fixed cells confirmed that SR proteins and snRNPs are present in the foci, and that there is a depletion of phosphorylated splicing factors in the regions that become immobilized. Morphological changes of nuclear speckles in living cells overexpressing GFP-Clk/STY(K190R) and phosphoepitope depletion in foci of such nuclear speckles strongly support the idea that phosphorylation of SR proteins by Clk/STY is one of the key events that results in recruitment of splicing factor complexes from nuclear speckles to sites of transcription. Furthermore, alterations in the phosphorylation state of SR proteins is highly correlated with IGC structural reorganization, and demonstrates an important link between structure and function in the mammalian cell nucleus.
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Materials and methods |
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Cell culture and transfection
A-431 cells were grown in DME containing high glucose (GIBCO BRL/ Life Technologies) supplemented with penicillin-streptomycin and 10% fetal bovine serum (Hyclone). Cells were seeded onto acid-washed coverslips in 35-mm petri dishes containing 2 ml DME, and attached cells were transiently transfected with 2 µg total DNA using FUGENE (Roche) according to manufacturer instructions. FLAG-Clk/STY was cotransfected with pTetON (CLONTECH Laboratories, Inc.), and expression of kinase was induced by addition of doxycycline (2.0 µg/ml). GFPKIAA fusion constructs were cotransfected with FLAG-Clk/STY + pTetOn 24 h before fixation, and doxycycline was added 1214 h before fixation. In all other experiments, cells were processed for immunofluorescence localization of nuclear speckle proteins 1416 h after transfection.
Immunofluorescence
Cells were rinsed briefly in PBS then fixed for 15 min in 2% formaldehyde in PBS (pH 7.4) or for 5 min in methanol (-20°C) for optimal penetration of IgM antibodies into nuclei. Cells were permeabilized in PBS +0.2% Triton X-100 + 0.5% goat serum, and primary antibodies were added for 1 h at room temperature: anti-FLAG M2 (Sigma-Aldrich; 10 µg/ml), SC35 (1:1000), 3C5 ascites (1:200), B" (1:200), mAb103 anti-SF2/ASF (1:15), mAb104 anti-SR (undiluted); M3 anti-pinin guinea pig serum (1:100), AC40 antiactin (Sigma-Aldrich; 1:100), 2H10 anti-lamin (undiluted), PABII (1:200); anti-snRNA m3G (1:40); ANA-N (fibrillarin, 1:10). Cells were rinsed in PBS + 0.5% goat serum, then secondary antispecies-specific antibodies (Jackson ImmunoResearch Laboratories) were added for 1 h at room temperature: goat antimouse (GAM) IgG1-Texas red (1:1,000), GAM IgG H+L Texas Red (1:500), GAM IgM-Cy5 (1:1,000), donkey antiguinea pig Cy5 (1:400), goat antirat IgG-fluorescein (1:1,200). Cells were examined using a ZEISS Axioplan 2i fluorescence microscope equipped with Chroma filters (Chroma Technology). OpenLab software (Improvision) was used to collect digital images.
TEM analysis
A-431 cells seeded onto gridded coverslips and transfected with GFP-Clk/STY were fixed for 15 min in 2% formaldehyde/0.5% glutaraldehyde in PBS (pH 7.4). Cells were rinsed in buffer A (PBS + 0.5% goat serum + 0.3 M glycine), then permeabilized for 20 min in PBS + 2% saponin. Anti-SC35 antibody was applied (1:1,000) in buffer B (PBS + 0.5% goat serum + 0.3 M glycine + 0.5% saponin) for 1 h at room temperature. Cells were rinsed in buffer B and Texas red GAM-IgG1 was applied (1:1,000) in buffer B. Cells were rinsed in buffer B, mounted, and sealed with rubber cement. The position of cells expressing GFP-Clk/STY and exhibiting completely disassembled SC35 nuclear speckles was documented and used later to relocate the cells for thin sectioning. Coverslips were processed as described (Huang et al., 1994); briefly, embedded cells were thin sectioned (100 nm), stained by the EDTA regressive method (Bernhard, 1969) and labeled with 3C5 antibody followed by 5 nm colloidal goldconjugated secondary antibody. Sections were examined using a Hitachi H-7000 TEM operated at 75 kilovolts.
Bromo-UTP incorporation
A-431 cells transfected with FLAG-Clk/STY were rinsed briefly in glycerol buffer (20 mM Tris HCl, pH 7.4, 5.0 mM MgCl2, 25% glycerol, 0.5 mM PMSF, and 0.5 mM EGTA) followed by permeabilization for 5 min in glycerol buffer supplemented with digitonin (20 µg/ml). Transcription buffer (100 mM KCl, 50 mM Tris HCl, pH 7.4, 5 mM MgCl2, 0.5 mM EGTA, 25% glycerol, 1.0 mM PMSF, 2.0 mM ATP, 0.5 mM GTP, 0.5 mM CTP, 0.2 mM bromo-UTP, 1.0 µg/ml RNAsin, 5.0 µg/ml digitonin) was added for 5 min at 37°C. Cells were rinsed in PBS then fixed in 2% formaldehyde in PBS (pH 7.4) for 15 min followed by methanol (-20°C) for 5 min. Triple localization of FLAG-Clk (M2), SR proteins (3C5), and bromo-UTP (rat anti-bromo, 1:30) was performed as described above.
RNA in situ hybridization for polyadenylated (polyA+) RNA
Cells transfected with FLAG-Clk/STY were fixed in 2% formaldehyde. Hybridization and detection of digoxygenin (DIG)-labeled oligo dT(50) probe was performed according to Huang et al. (1994). FLAG-Clk/STY was detected with M2 anti-FLAG antibody (Sigma-Aldrich) followed by Texas red GAM IgG1.
Nick-translation of ß-globin genomic probe
ß-globin genomic DNA was subcloned into pBluescript (Stratagene). 2 µg DNA was nick translated in buffer (50 mM Tris HCl, pH 8.0; 5.0 mM MgCl2; 0.05 mg/ml BSA; 0.001 M ß-mercaptoethanol; 0.5 mM each dATP, dGTP, and dCTP; 0.125 mM dTTP; 0.375 mM DIG-11-dUTP) containing 0.01 mg/ml DNaseI and 10 U E. coli DNA polymerase for 2 h at 16°C to obtain fragments of 300500 bp. The probe was precipitated, resuspended in 20 µl water, and 2 µl of this suspension was used per hybridization sample.
RNA FISH
A-431 cells were stably transfected with ß-globin genomic DNA using FUGENE and selected in 0.1 mg/ml G-418. Two stable clones were used to examine splicing factor recruitment and splicing of the reporter pre-mRNA. GFP-Clk/STY was transfected by electroporation optimized for A-431 cells. Trypsinized cells were washed in PBS, resuspended in 500 µl ice-cold cytomix buffer (10 mM K2HPO4, 10 mM KH2PO4, 25 mM Hepes, 120 mM KCl, 0.15 mM CaCl2, 5 mM MgCl2, 2 mM EGTA, 5 mM glutathione, and 2 mM ATP; pH 7.6), and added to cuvettes containing 12 µg GFP-Clk/STY. After a 5 min incubation on ice, cells were electroporated (380 mV, 950 µF), transferred to 6 ml culture medium, and plated 2 ml/coverslip coated with fibronectin (Sigma-Aldrich) to improve A-431 electroporation efficiency (Hashino et al., 1997). 16 h after transfection, cells were processed for colocalization of SC35 with ß-globin pre-mRNA. Cells were fixed for 15 min in 2% formaldehyde, permeabilized 5 min in PBS + 0.5% Triton X-100, washed 3 times for 5 min each in PBS + 0.02% BSA (New England BioLabs, Inc.), incubated in anti-SC35 (1:1,000) + 0.1% BSA for 1 h at room temperature, washed 3 times for 5 min each in PBS, and incubated in Cy5-conjugated GAM IgG (1:800; Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. Cells were washed 3 times for 5 min each in PBS and fixed for 5 min in 2% formaldehyde followed by washing 3 times for 5 min each in PBS and 10 min in 2XSSC. Denatured nick-translated DIG-11-dUTPlabeled ß-globin probe (200 ng) was added in 50% deionized formamide, 10% dextran sulfate, 1 mg/ml yeast tRNA and 2XSSC, and coverslips were inverted onto slides and sealed with rubber cement for overnight hybridization at 37°C in a humidified chamber. Cells were washed 30 min in 50% formamide/2XSSC at 37°C, then 30 min each in 2XSSC and 1XSSC. Probe was detected with sheep anti-DIG Fab fragments (1:300; Roche) followed by donkey antisheep Texas red (1:600; Jackson ImmunoResearch Laboratories).
To detect splicing of ß-globin pre-mRNA, 30-mer oligonucleotides conjugated with a single Texas red molecule at the 5' end (GIBCO BRL) were designed to hybridize to intron 2 (i2: 5'-gacttccacactgatgcaatcattcgtctg-3') or to the splice junction between exons 2 and 3 (e2/3: 5'-cacgttgcccaggagcctgaagttctcagg-3'). Cells were transfected with GFP-Clk/STY by electroporation as described above. Cells were extracted in CSK buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM Pipes, pH 6.8) supplemented with 0.5% Triton X-100 and 2 mM vanadyl ribonucleoside complex, and hybridization of oligo probes was performed in 25% deionized formamide, 10% dextran sulfate, 1 mg/ml yeast tRNA, and 2XSSC for 3 h at 37°C. Cells were washed for 30 min in 25% formamide/2XSSC at 37°C then 30 min in 2XSSC. Hybridization signals were scored in untransfected versus transfected cells. Multiple focal planes were examined to confirm the absence of signal in cells without intact nuclear speckles.
Live cell microscopy
Attached cells were transfected with GFP-Clk/STY using FUGENE as described above. The cells were transferred 4 h after transfection to an FCS2 live-cell chamber (Bioptechs) mounted onto the stage of an Olympus IX70 inverted fluorescence microscope (Olympus) and kept at 37°C in L-15 medium containing 10% FBS and without phenol red. Time-lapse images acquired with a 100x 1.4 NA heated objective lens were captured with a peltier-cooled IMAGO CCD camera using an SVGA interline chip (1,280 x 1,024) with a pixel size of 6.7 x 6.7 µm (Till Photonics) as soon as nuclear expression was initially detected (at 6.0 h). For GFP-Clk/STY(K190R), a sequence of 200 exposures (350 ms each) was recorded every 30 min.
Online supplemental material
Videos corresponding to Fig. 8 are presented. Image sequences were acquired using TillVision software (Till Photonics) and animated using QuickTime software. For each video a sequence of 200 images (350 ms each) was taken. Video speed is five times faster than real time. Video 1 shows nuclear speckle dynamics in a cell overexpressing GFP-Clk/STY(K190R) at 6.0 h posttransfection. GFP-Clk/STY localizes to nuclear speckles and exhibits rapid movement in and out of the speckles. Focal accumulations of GFP-Clk/STY are seen on several speckles. Video 2 shows the same cell at 6.5 h posttransfection. Although all of the nuclear speckles exhibit foci at this stage, nuclear speckle dynamics outside of the foci are largely unaffected. Video 3 shows the same cell at 7.0 h posttransfection. As multiple foci form on each nuclear speckle, they appear paired and larger regions of the speckles become immobilized. Video 4 shows the same cell at 7.5 h posttransfection. The nuclear speckles are almost completely immobilized. Videos are available at http://www.jcb.org/cgi/content/full/jcb.200107017/DC1.
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Footnotes |
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* Abbreviations used in this paper: Clk, cdc2-like kinase; DIG, digoxygenin; GFP, green fluorescent protein; IGC, interchromatin granule cluster; PF, perichromatin fibril; RS, arginine-serine rich; SR, serine-arginine; TEM, transmission electron microscopy.
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Acknowledgments |
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P. Sacco-Bubulya is funded by a postdoctoral fellowship from NIH/NIGMS. D.L. Spector is supported by NIH/NIGMS 42694.
Submitted: 5 July 2001
Revised: 10 December 2001
Accepted: 14 December 2001
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References |
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Alahari, S., H. Schmidt, and N. Kaufer. 1993. The fission yeast prp4+ gene involved in pre-mRNA splicing codes for a predicted serine/threonine kinase and is essential for growth. Nucleic Acids Res. 21:40794083.[Abstract]
Ben-David, Y., K. Letwin, L. Tannock, A. Bernstein, and T. Pawson. 1991. A mammalian protein kinase with potential for serine/threonine and tyrosine phosphorylation is related to cell cycle regulators. EMBO J. 10:317325.[Abstract]
Boronenkov, I.V., J.C. Loijens, M. Umeda, and R.A. Anderson. 1998. Phosphoinositide signaling pathways in nuclei are associated with nuclear speckles containing pre-mRNA processing factors. Mol. Biol. Cell. 9:35473560.
Bregman, D.B., L. Du, S. van der Zee, and S.L. Warren. 1995. Transcription-dependent redistribution of the large subunit of RNA polymerase II to discrete nuclear domains. J. Cell Biol. 129:287298.[Abstract]
Carmo-Fonseca, M., R. Pepperkok, M.T. Carvalho, and A.I. Lamond. 1992. Transcription-dependent colocalization of the U1, U2, U4/U6 and U5 snRNPs in coiled bodies. J. Cell Biol. 117:114.[Abstract]
Cmarko, D., P.J. Verschure, T.E. Martin, M.E. Dahmus, S. Krause, X.D. Fu, R. van Driel, and S. Fakan. 1999. Ultrastructural analysis of transcription and splicing in the cell nucleus after bromo-UTP microinjection. Mol. Biol. Cell. 10:211223.
Colwill, K., L.L. Feng, J.M. Yeakley, G.D. Gish, J.F. Cáceres, T. Pawson, and X.-D. Fu. 1996a. SRPK1 and Clk/Sty protein kinases show distinct substrate specificities for serine/arginine-rich splicing factors. J. Biol. Chem. 271:2456924575.
Colwill, K., T. Pawson, B. Andrews, J. Prasad, J.L. Manley, J.C. Bell, and P.I. Duncan. 1996b. The Clk/Sty protein kinase phosphorylates splicing factors and regulates their intranuclear distribution. EMBO J. 15:265275.[Abstract]
Duncan, P.I., D.F. Stojdl, R.M. Marius, and J.C. Bell. 1997. In vivo regulation of alternative pre-mRNA splicing by the Clk1 protein kinase. Mol. Cell. Biol. 17:59966001.[Abstract]
Eils, R., D. Gerlich, W. Tvarusko, D.L. Spector, and T. Misteli. 2000. Quantitative imaging of pre-mRNA splicing factors in living cells. Mol. Biol. Cell. 11:413418.
Fakan, S., and E. Puvion. 1980. The ultrastructural visualization of nucleolar and extranucleolar RNA synthesis and distribution. Int. Rev. Cytol. 65:255299.[Medline]
Gall, J.G., M. Bellini, Z. Wu, and C. Murphy. 1999. Assembly of the nuclear transcription and processing machinery: Cajal bodies (coiled bodies) and transcriptosomes. Mol. Biol. Cell. 10:43854402.
Habets, W.J., M.H. Hoet, B.A.W. DeJong, A. VanDer Kemp, and W.J. Van Venrooij. 1986. Mapping of B cell epitopes on small nuclear ribonucleoproteins that react with human autoantibodies as well as with experimentally induced mouse monoclonal antibodies. J. Immunol. 143:25602566.
Hashino, K., H. Matsushita, and I. Kato. 1997. Effects of fibronectin fragments on DNA transfection into mammalian cells by electroporation. J. Biochem. 122:490493.[Abstract]
Howell, B.W., D.E. Afar, J. Lew, E.M. Douville, P.L. Icely, D.A. Gray, and J.C. Bell. 1991. STY, a tyrosine-phosphorylating enzyme with sequence homology to serine/threonine kinases. Mol. Cell. Biol. 11:568572.[Medline]
Huang, S., T.J. Deerinck, M.H. Ellisman, and D.L. Spector. 1994. In vivo analysis of the stability and transport of nuclear poly(A)+ RNA. J. Cell Biol. 126:877899.[Abstract]
Huang, S., and D.L. Spector. 1996. Intron-dependent recruitment of pre-mRNA splicing factors to sites of transcription. J. Cell Biol. 131:719732.
Jagatheesan, G., S. Thanumalayan, B. Muralikrishna, N. Rangaraj, A.A. Karande, and V.K. Parnaik. 1999. Colocalization of intranuclear lamin foci with RNA splicing factors. J. Cell Sci. 112:46514661.
Johnson, K.W., and K.A. Smith. 1991. Molecular cloning of a novel human cdc2/CDC28-like protein kinase. J. Biol. Chem. 266:34023407.
Kojima, T., T. Zama, K. Wada, H. Onogi, and M. Hagiwara. 2001. Cloning of human PRP4 reveals interaction with Clk1. J. Biol. Chem. 276:3224732256.
Kruhlak, M.J., M.A. Lever, W. Fischle, E. Verdin, D.P. Bazett-Jones, and M.J. Hendzel. 2000. Reduced mobility of the alternate splicing factor (ASF) through the nucleoplasm and steady state speckle compartments. J. Cell Biol. 150:4151.
Lamond, A.I., and W.C. Earnshaw. 1998. Structure and function in the nucleus. Science. 280:547553.
Lee, K., C. Du, M. Horn, and L. Rabinow. 1996. Activity and autophosphorylation of LAMMER protein kinases. J. Biol. Chem. 271:2729927303.
Li, Q., H. Imataka, S. Morino, G.W. Rogers, Jr., N.J. Richter-Cook, W.C. Merrick, and N. Sonenberg. 1999. Eukaryotic translation initiation factor 4AIII (eIF4AIII) is functionally distinct from eIF4AI and eIF4AII. Mol. Cell. Biol. 19:73367346.
Mintz, P.J., S.D. Patterson, A.F. Neuwald, C.S. Spahr, and D.L. Spector. 1999. Purification and biochemical characterization of interchromatin granule clusters. EMBO J. 18:43084320.
Nakayasu, H., and K. Ueda. 1984. Small nuclear RNA-protein complex anchors on the actin filaments in bovine lymphocyte nuclear matrix. Cell Struct. Funct. 9:317325.[Medline]
O'Keefe, R.T., A. Mayeda, C.L. Sadowski, A.R. Krainer, and D.L. Spector. 1994. Disruption of pre-mRNA splicing in-vivo results in reorganization of splicing factors. J. Cell Biol. 124:249260.[Abstract]
Ouyang, P., and S.P. Sugrue. 1996. Characterization of pinin, a novel protein associated with the desmosome-intermediate filament complex. J. Cell Biol. 135:10271042.[Abstract]
Percipalle, P., J. Zhao, B. Pope, A. Weeds, U. Lindberg, and B. Daneholt. 2001. Actin bound to the heterogeneous nuclear ribonucleoprotein hrp36 is associated with balbiani ring mRNA from the gene to polysomes. J. Cell Biol. 153:229236.
Prasad, J., K. Colwill, T. Pawson, and J.L. Manley. 1999. The protein kinase Clk/Sty directly modulates SR protein activity: both hyper- and hypophosphorylation inhibit splicing. Mol. Cell. Biol. 19:69917000.
Roth, M.B., C. Murphy, and J.G. Gall. 1990. A monoclonal antibody that recognizes a phosphorylated epitope stains lampbrush chromosomes and small granules in the amphibian germinal vesicle. J. Cell Biol. 111:22172223.[Abstract]
Spector, D.L. 1990. Higher order nuclear organization: three-dimensional distribution of small nuclear ribonucleoprotein particles. Proc. Natl. Acad. Sci. USA. 87:147151.[Abstract]
Spector, D.L., G. Lark, and S. Huang. 1992. Differences in snRNP localization between transformed and nontransformed cells. Mol. Biol. Cell. 3:555569.[Abstract]
Spector, D.L., R.T. O'Keefe, and L.F. Jiménez-García. 1993. Dynamics of transcription and pre-mRNA splicing within the mammalian cell nucleus. Cold Spring Harb. Symp. Quant. Biol. 58:799805.[Medline]
Turner, B.M., and L. Franchi. 1987. Identification of protein antigens associated with the nuclear matrix and with clusters of interchromatin granules in both interphase and mitotic cells. J. Cell Sci. 87:269282.[Abstract]
Wang, H.-Y., W. Lin, J.A. Dyck, J.M. Yeakley, Z. Songyang, L.C. Cantley, and X.-D. Fu. 1998. SRPK2: A differentially expressed SR protein-specific kinase involved in mediating the interaction and localization of pre-mRNA splicing factors in mammalian cells. J. Cell Biol. 140:737-750
Wei, X., S. Somanathan, J. Samarabandu, and R. Berezney. 1999. Three-dimensional visualization of transcription sites and their association with splicing factor-rich nuclear speckles. J. Cell Biol. 146:543558.
Weinstein, D.C., E. Honore, and A. Hemmati-Brivanlou. 1997. Epidermal induction and inhibition of neural fate by translation initiation factor 4AIII. Development. 124:42354242.
Yeakley, J.M., H. Tronchere, J. Olesen, J.A. Dyck, H.Y. Wang, and X.D. Fu. 1999. Phosphorylation regulates in vivo interaction and molecular targeting of serine/arginine-rich pre-mRNA splicing factors. J. Cell Biol. 145:447455.
Zhao, K., W. Wang, O.J. Rando, Y. Xue, K. Swiderek, A. Kuo, and G.R. Crabtree. 1988. Rapid and phosphoinositol-dependent binding of the SWI/SNF-like BAF complex to chromatin after T lymphocyte receptor signaling. Cell. 95:625636.