scholarly journals Serine Phosphorylation of SR Proteins Is Required for Their Recruitment to Sites of Transcription In Vivo

1998 ◽  
Vol 143 (2) ◽  
pp. 297-307 ◽  
Author(s):  
Tom Misteli ◽  
Javier F. Cáceres ◽  
Jade Q. Clement ◽  
Adrian R. Krainer ◽  
Miles F. Wilkinson ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Active Sites ◽  
Rna Binding ◽  
Sr Proteins ◽  
Mrna Splicing ◽  
Serine Phosphorylation ◽  
Splicing Factors ◽  
Nuclear Speckles

Expression of most RNA polymerase II transcripts requires the coordinated execution of transcription, splicing, and 3′ processing. We have previously shown that upon transcriptional activation of a gene in vivo, pre-mRNA splicing factors are recruited from nuclear speckles, in which they are concentrated, to sites of transcription (Misteli, T., J.F. Cáceres, and D.L. Spector. 1997. Nature. 387:523–527). This recruitment process appears to spatially coordinate transcription and pre-mRNA splicing within the cell nucleus. Here we have investigated the molecular basis for recruitment by analyzing the recruitment properties of mutant splicing factors. We show that multiple protein domains are required for efficient recruitment of SR proteins from nuclear speckles to nascent RNA. The two types of modular domains found in the splicing factor SF2/ ASF exert distinct functions in this process. In living cells, the RS domain functions in the dissociation of the protein from speckles, and phosphorylation of serine residues in the RS domain is a prerequisite for this event. The RNA binding domains play a role in the association of splicing factors with the target RNA. These observations identify a novel in vivo role for the RS domain of SR proteins and suggest a model in which protein phosphorylation is instrumental for the recruitment of these proteins to active sites of transcription in vivo.

scholarly journals A conserved epitope on a subset of SR proteins defines a larger family of Pre-mRNA splicing factors.

1995 ◽  
Vol 129 (4) ◽  
pp. 899-908 ◽  
Author(s):  
K M Neugebauer ◽  
J A Stolk ◽  
M B Roth
Keyword(s):  
Rna Polymerase Ii ◽  
Active Sites ◽  
Nuclear Extract ◽  
Sr Proteins ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Structural Element ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

The removal of introns from eukaryotic pre-mRNA occurs in a large ribonucleoprotein complex called the spliceosome. We have generated a monoclonal antibody (mAb 16H3) against four of the family of six SR proteins, known regulators of splice site selection and spliceosome assembly. In addition to the reactive SR proteins, SRp20, SRp40, SRp55, and SRp75, mAb 16H3 also binds approximately 20 distinct nuclear proteins in human, frog, and Drosophila extracts, whereas yeast do not detectably express the epitope. The antigens are shown to be nuclear, nonnucleolar, and concentrated at active sites of RNA polymerase II transcription which suggests their involvement in pre-mRNA processing. Indeed, most of the reactive proteins observed in nuclear extract are detected in spliceosomes (E and/or B complex) assembled in vitro, including the U1 70K component of the U1 small nuclear ribonucleoprotein particle and both subunits of U2AF. Interestingly, the 16H3 epitope was mapped to a 40-amino acid polypeptide composed almost exclusively of arginine alternating with glutamate and aspartate. All of the identified antigens, including the human homolog of yeast Prp22 (HRH1), contain a similar structural element characterized by arginine alternating with serine, glutamate, and/or aspartate. These results indicate that many more spliceosomal components contain such arginine-rich domains. Because it is conserved among metazoans, we propose that the "alternating arginine" domain recognized by mAb 16H3 may represent a common functional element of pre-mRNA splicing factors.

scholarly journals Serine/threonine phosphatase 1 modulates the subnuclear distribution of pre-mRNA splicing factors.

1996 ◽  
Vol 7 (10) ◽  
pp. 1559-1572 ◽  
Author(s):  
T Misteli ◽  
D L Spector
Keyword(s):  
Rna Polymerase Ii ◽  
Mammalian Cell ◽  
Cell Nucleus ◽  
Nuclear Extract ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Cell Nuclei ◽  
Nuclear Speckles ◽  
Permeabilized Cells

HeLa cell nuclei were permeabilized and reconstituted with nuclear extract to identify soluble nuclear factors which play a role in the organization of pre-mRNA splicing factors in the mammalian cell nucleus. Permeabilized nuclei reconstituted with nuclear extract were active in transcription and DNA replication and nuclear speckles containing pre-mRNA splicing factors were maintained over several hours independent of soluble nuclear components. The characteristic rounding up of nuclear speckles in response to inhibition of RNA polymerase II seen in vivo was reproduced in permeabilized cells and was strictly dependent on a catalytic activity present in the nuclear extract. By inhibitor titration experiments and sensitivity to inhibitor 2, this activity was identified as a member of the serine/threonine protein phosphatase 1 family (PP1). Interference with PP1 activity affected the distribution of pre-mRNA splicing factors in transcriptionally active, permeabilized cells, and excess PP1 activity caused increased dephosphorylation of SR proteins in nuclear speckles. These data show that the dynamic reorganization of the mammalian cell nucleus can be studied in permeabilized cells and that PP1 is involved in the rounding up of speckles as well as the overall organization of pre-mRNA splicing factors in the mammalian cell nucleus.

scholarly journals A Functional Interaction between the Carboxy-Terminal Domain of RNA Polymerase II and Pre-mRNA Splicing

1997 ◽  
Vol 136 (1) ◽  
pp. 5-18 ◽  
Author(s):  
Lei Du ◽  
Stephen L. Warren
Keyword(s):  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Functional Interaction ◽  
Carboxy Terminal Domain ◽  
Localization Signal ◽  
Nuclear Domains ◽  
Carboxy Terminal

In the preceding study we found that Sm snRNPs and SerArg (SR) family proteins co-immunoprecipitate with Pol II molecules containing a hyperphosphorylated CTD (Kim et al., 1997). The association between Pol IIo and splicing factors is maintained in the absence of pre-mRNA, and the polymerase need not be transcriptionally engaged (Kim et al., 1997). The latter findings led us to hypothesize that a phosphorylated form of the CTD interacts with pre-mRNA splicing components in vivo. To test this idea, a nested set of CTD-derived proteins was assayed for the ability to alter the nuclear distribution of splicing factors, and to interfere with splicing in vivo. Proteins containing heptapeptides 1-52 (CTD52), 1-32 (CTD32), 1-26 (CTD26), 1-13 (CTD13), 1-6 (CTD6), 1-3 (CTD3), or 1 (CTD1) were expressed in mammalian cells. The CTD-derived proteins become phosphorylated in vivo, and accumulate in the nucleus even though they lack a conventional nuclear localization signal. CTD52 induces a selective reorganization of splicing factors from discrete nuclear domains to the diffuse nucleoplasm, and significantly, it blocks the accumulation of spliced, but not unspliced, human β-globin transcripts. The extent of splicing factor disruption, and the degree of inhibition of splicing, are proportional to the number of heptapeptides added to the protein. The above results indicate a functional interaction between Pol II's CTD and pre-mRNA splicing.

scholarly journals Targeting of U2AF65 to Sites of Active Splicing in the Nucleus

1997 ◽  
Vol 137 (5) ◽  
pp. 975-987 ◽  
Author(s):  
Margarida Gama-Carvalho ◽  
Randy D. Krauss ◽  
Lijian Chiang ◽  
Juan Valcárcel ◽  
Michael R. Green ◽  
...  
Keyword(s):  
Transient Expression ◽  
Rna Binding ◽  
Splicing Factors ◽  
Deletion Mutants ◽  
Adenovirus Infection ◽  
Nuclear Speckles ◽  
Adenoviral Infection ◽  
Transcription Inhibitors ◽  
G1 Cells

U2AF65 is an essential splicing factor that promotes binding of U2 small nuclear (sn)RNP at the pre-mRNA branchpoint. Here we describe a novel monoclonal antibody that reacts specifically with U2AF65. Using this antibody, we show that U2AF65 is diffusely distributed in the nucleoplasm with additional concentration in nuclear speckles, which represent subnuclear compartments enriched in splicing snRNPs and other splicing factors. Furthermore, transient expression assays using epitope-tagged deletion mutants of U2AF65 indicate that targeting of the protein to nuclear speckles is not affected by removing either the RNA binding domain, the RS domain, or the region required for interaction with U2AF35. The association of U2AF65 with speckles persists during mitosis, when transcription and splicing are downregulated. Moreover, U2AF65 is localized to nuclear speckles in early G1 cells that were treated with transcription inhibitors during mitosis, suggesting that the localization of U2AF65 in speckles is independent of the presence of pre-mRNA in the nucleus, which is consistent with the idea that speckles represent storage sites for inactive splicing factors. After adenovirus infection, U2AF65 redistributes from the speckles and is prefferentially detected at sites of viral transcription. By combining adenoviral infection with transient expression of deletion mutants, we show a specific requirement of the RS domain for recruitment of U2AF65 to sites of active splicing in the nucleus. This suggests that interactions involving the RS region of U2AF65 may play an important role in targeting this protein to spliceosomes in vivo.

scholarly journals The WW Domain-Containing Proteins Interact with the Early Spliceosome and Participate in Pre-mRNA Splicing In Vivo

2004 ◽  
Vol 24 (20) ◽  
pp. 9176-9185 ◽  
Author(s):  
Kai-Ti Lin ◽  
Ruei-Min Lu ◽  
Woan-Yuh Tarn
Keyword(s):  
Rna Polymerase Ii ◽  
Nuclear Extract ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Ww Domain ◽  
Ww Domains ◽  
Small Nuclear Ribonucleoprotein

ABSTRACT A growing body of evidence supports the coordination of mRNA synthesis and its subsequent processing events. Nuclear proteins harboring both WW and FF protein interaction modules bind to splicing factors as well as RNA polymerase II and may serve to link transcription with splicing. To understand how WW domains coordinate the assembly of splicing complexes, we used glutathione S-transferase fusions containing WW domains from CA150 or FBP11 in pull-down experiments with HeLa cell nuclear extract. The WW domains associate preferentially with the U2 small nuclear ribonucleoprotein and with splicing factors SF1, U2AF, and components of the SF3 complex. Accordingly, WW domain-associating factors bind to the 3′ part of a pre-mRNA to form a pre-spliceosome-like complex. We performed both in vitro and in vivo splicing assays to explore the role of WW/FF domain-containing proteins in this process. However, although CA150 is associated with the spliceosome, it appears to be dispensable for splicing in vitro. Nevertheless, in vivo depletion of CA150 substantially reduced splicing efficiency of a reporter pre-mRNA. Moreover, overexpression of CA150 fragments containing both WW and FF domains activated splicing and modulated alternative exon selection, probably by facilitating 3′ splice site recognition. Our results suggest an essential role of WW/FF domain-containing factors in pre-mRNA splicing that likely occurs in concert with transcription in vivo.

scholarly journals Differential effects of hyperphosphorylation on splicing factor SRp55

2003 ◽  
Vol 371 (3) ◽  
pp. 937-945 ◽  
Author(s):  
Ming-Chih LAI ◽  
Ru-Inn LIN ◽  
Woan-Yuh TARN
Keyword(s):  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Sr Proteins ◽  
Binding Activity ◽  
Protein Family ◽  
Nuclear Speckles ◽  
Sr Protein ◽  
Rich Domain ◽  
First Time ◽  
Amino Acid Segment

Members of the serine/arginine-rich (SR) protein family play an important role in both constitutive and regulated splicing of precursor mRNAs. Phosphorylation of the arginine/serine dipeptide-rich domain (RS domain) can modulate the activity and the subcellular localization of SR proteins. However, whether the SR protein family members are individually regulated and how this is achieved remain unclear. In this report we show that 5,6-dichloro-1β-d-ribofuranosyl-benzimidazole (DRB), an inhibitor of RNA polymerase II-dependent transcription, specifically induced hyperphosphorylation of SRp55 but not that of any other SR proteins tested. Hyperphosphorylation of SRp55 occurs at the RS domain and appears to require the RNA-binding activity. Upon DRB treatment, hyperphosphorylated SRp55 relocates to enlarged nuclear speckles. Intriguingly, SRp55 is specifically targeted for degradation by the proteasome upon overexpression of the SR protein kinase Clk/Sty. Although a destabilization signal is mapped within the C-terminal 43-amino acid segment of SRp55, its adjacent lysine/serine-rich RS domain is nevertheless critical for the Clk/Sty-mediated degradation. We report for the first time that SRp55 can be hyperphosphorylated under different circumstances whereby its fate is differentially influenced.

scholarly journals Hypophosphorylated SR splicing factors transiently localize around active nucleolar organizing regions in telophase daughter nuclei

2004 ◽  
Vol 167 (1) ◽  
pp. 51-63 ◽  
Author(s):  
Paula A. Bubulya ◽  
Kannanganattu V. Prasanth ◽  
Thomas J. Deerinck ◽  
Daniel Gerlich ◽  
Joel Beaudouin ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Sr Proteins ◽  
Splicing Factors ◽  
Nuclear Speckles ◽  
Sr Protein ◽  
Nucleolar Organizing Regions ◽  
Transcription Sites ◽  
Nuclear Regions ◽  
Rna Polymerase Ii Transcription

Upon completion of mitosis, daughter nuclei assemble all of the organelles necessary for the implementation of nuclear functions. We found that upon entry into daughter nuclei, snRNPs and SR proteins do not immediately colocalize in nuclear speckles. SR proteins accumulated in patches around active nucleolar organizing regions (NORs) that we refer to as NOR-associated patches (NAPs), whereas snRNPs were enriched at other nuclear regions. NAPs formed transiently, persisting for 15–20 min before dissipating as nuclear speckles began to form in G1. In the absence of RNA polymerase II transcription, NAPs increased in size and persisted for at least 2 h, with delayed localization of SR proteins to nuclear speckles. In addition, SR proteins in NAPs are hypophosphorylated, and the SR protein kinase Clk/STY colocalizes with SR proteins in NAPs, suggesting that phosphorylation releases SR proteins from NAPs and their initial target is transcription sites. This work demonstrates a previously unrecognized role of NAPs in splicing factor trafficking and nuclear speckle biogenesis.

scholarly journals SRSF1 regulates the assembly of pre-mRNA processing factors in nuclear speckles

2012 ◽  
Vol 23 (18) ◽  
pp. 3694-3706 ◽  
Author(s):  
Vidisha Tripathi ◽  
David Y. Song ◽  
Xinying Zong ◽  
Sergey P. Shevtsov ◽  
Stephen Hearn ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Cell Nucleus ◽  
Interphase Nucleus ◽  
Sr Proteins ◽  
Mrna Processing ◽  
Splicing Factors ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Processing Factors

The mammalian cell nucleus is compartmentalized into nonmembranous subnuclear domains that regulate key nuclear functions. Nuclear speckles are subnuclear domains that contain pre-mRNA processing factors and noncoding RNAs. Many of the nuclear speckle constituents work in concert to coordinate multiple steps of gene expression, including transcription, pre-mRNA processing and mRNA transport. The mechanism that regulates the formation and maintenance of nuclear speckles in the interphase nucleus is poorly understood. In the present study, we provide evidence for the involvement of nuclear speckle resident proteins and RNA components in the organization of nuclear speckles. SR-family splicing factors and their binding partner, long noncoding metastasis-associated lung adenocarcinoma transcript 1 RNA, can nucleate the assembly of nuclear speckles in the interphase nucleus. Depletion of SRSF1 in human cells compromises the association of splicing factors to nuclear speckles and influences the levels and activity of other SR proteins. Furthermore, on a stably integrated reporter gene locus, we demonstrate the role of SRSF1 in RNA polymerase II–mediated transcription. Our results suggest that SR proteins mediate the assembly of nuclear speckles and regulate gene expression by influencing both transcriptional and posttranscriptional activities within the cell nucleus.

scholarly journals Disassembly of interchromatin granule clusters alters the coordination of transcription and pre-mRNA splicing

2002 ◽  
Vol 156 (3) ◽  
pp. 425-436 ◽  
Author(s):  
Paula Sacco-Bubulya ◽  
David L. Spector
Keyword(s):  
Mammalian Cell ◽  
Protein Interactions ◽  
Sr Proteins ◽  
Mrna Splicing ◽  
Cell Nuclei ◽  
Nuclear Speckles ◽  
Sr Protein ◽  
Interchromatin Granule Clusters ◽  
Interchromatin Granule

To examine the involvement of interchromatin granule clusters (IGCs) in transcription and pre-mRNA splicing in mammalian cell nuclei, the serine-arginine (SR) protein kinase cdc2-like kinase (Clk)/STY was used as a tool to manipulate IGC integrity in vivo. Both immunofluorescence and transmission electron microscopy analyses of cells overexpressing Clk/STY indicate that IGC components are completely redistributed to a diffuse nuclear localization, leaving no residual structure. Conversely, overexpression of a catalytically inactive mutant, Clk/STY(K190R), causes retention of hypophosphorylated SR proteins in nuclear speckles. Our data suggest that the protein–protein interactions responsible for the clustering of interchromatin granules are disrupted when SR proteins are hyperphosphorylated and stabilized when SR proteins are hypophosphorylated. Interestingly, cells without intact IGCs continue to synthesize nascent transcripts. However, both the accumulation of splicing factors at sites of pre-mRNA synthesis as well as pre-mRNA splicing are dramatically reduced, demonstrating that IGC disassembly perturbs coordination between transcription and pre-mRNA splicing in mammalian cell nuclei.

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