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 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 In vitro analysis of a transcription termination site for RNA polymerase II.

1990 ◽  
Vol 10 (11) ◽  
pp. 5782-5795 ◽  
Author(s):  
D K Wiest ◽  
D K Hawley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Transcription Unit ◽  
Dependent Manner ◽  
Wide Range ◽  
Vitro Analysis

Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.

scholarly journals PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle

1989 ◽  
Vol 9 (9) ◽  
pp. 3710-3719
Author(s):  
J Banroques ◽  
J N Abelson
Keyword(s):  
Essential Role ◽  
Mrna Splicing ◽  
Ribonucleoprotein Particle ◽  
Specific Antibodies ◽  
Small Nuclear Ribonucleoprotein ◽  
Assembly Pathway ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle

The Saccharomyces cerevisiae prp mutants (prp2 through prp11) are known to be defective in pre-mRNA splicing at nonpermissive temperatures. We have sequenced the PRP4 gene and shown that it encodes a 52-kilodalton protein. We obtained PRP4 protein-specific antibodies and found that they inhibited in vitro pre-mRNA splicing, which confirms the essential role of PRP4 in splicing. Moreover, we found that PRP4 is required early in the spliceosome assembly pathway. Immunoprecipitation experiments with anti-PRP4 antibodies were used to demonstrate that PRP4 is a protein of the U4/U6 small nuclear ribonucleoprotein particle (snRNP). Furthermore, the U5 snRNP could be immunoprecipitated through snRNP-snRNP interactions in the large U4/U5/U6 complex.

scholarly journals Human Cancer-Associated Mutations of SF3B1 Lead to a Splicing Modification of Its Own RNA

Cancers ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 652
Author(s):  
Tiffany Bergot ◽  
Eric Lippert ◽  
Nathalie Douet-Guilbert ◽  
Séverine Commet ◽  
Laurent Corcos ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Cancer ◽  
Myeloid Cell ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Small Nuclear Ribonucleoprotein ◽  
Genes Encoding ◽  
Protein Isoform ◽  
Nuclear Ribonucleoprotein

Deregulation of pre-mRNA splicing is observed in many cancers and hematological malignancies. Genes encoding splicing factors are frequently mutated in myelodysplastic syndromes, in which SF3B1 mutations are the most frequent. SF3B1 is an essential component of the U2 small nuclear ribonucleoprotein particle that interacts with branch point sequences close to the 3’ splice site during pre-mRNA splicing. SF3B1 mutations mostly lead to substitutions at restricted sites in the highly conserved HEAT domain, causing a modification of its function. We found that SF3B1 was aberrantly spliced in various neoplasms carrying an SF3B1 mutation, by exploring publicly available RNA sequencing raw data. We aimed to characterize this novel SF3B1 transcript, which is expected to encode a protein with an insertion of eight amino acids in the H3 repeat of the HEAT domain. We investigated the splicing proficiency of this SF3B1 protein isoform, in association with the most frequent mutation (K700E), through functional complementation assays in two myeloid cell lines stably expressing distinct SF3B1 variants. The yeast Schizosaccharomyces pombe was also used as an alternative model. Insertion of these eight amino acids in wild-type or mutant SF3B1 (K700E) abolished SF3B1 essential function, highlighting the crucial role of the H3 repeat in the splicing function of SF3B1.

scholarly journals Interaction between the Negative Regulator of Splicing Element and a 3′ Splice Site: Requirement for U1 Small Nuclear Ribonucleoprotein and the 3′ Splice Site Branch Point/Pyrimidine Tract

1999 ◽  
Vol 73 (3) ◽  
pp. 2394-2400 ◽  
Author(s):  
Craig R. Cook ◽  
Mark T. McNally
Keyword(s):  
Branch Point ◽  
Splice Site ◽  
Rna Splicing ◽  
Nuclear Extract ◽  
Negative Regulator ◽  
Rous Sarcoma ◽  
Small Nuclear Ribonucleoprotein ◽  
A Minor

ABSTRACT The negative regulator of splicing (NRS) from Rous sarcoma virus suppresses viral RNA splicing and is one of several ciselements that account for the accumulation of large amounts of unspliced RNA for use as gag-pol mRNA and progeny virion genomic RNA. The NRS can also inhibit splicing of heterologous introns in vivo and in vitro. Previous data showed that the splicing factors SF2/ASF and U1, U2, and U11 small nuclear ribonucleoproteins (snRNPs) bind the NRS, and a correlation was established between SF2/ASF and U11 binding and activity, suggesting that these factors are important for function. These observations, and the finding that a large spliceosome-like complex (NRS-C) assembles on NRS RNA in nuclear extract, led to the proposal that the NRS is recognized as a minor-class 5′ splice site. One model to explain NRS splicing inhibition holds that the NRS interacts nonproductively with and sequesters U2-dependent 3′ splice sites. In this study, we provide evidence that the NRS interacts with an adenovirus 3′ splice site. The interaction was dependent on the integrity of the branch point and pyrimidine tract of the 3′ splice site, and it was sensitive to a mutation that was previously shown to abolish U11 snRNP binding and NRS function. However, further mutational analyses of NRS sequences have identified a U1 binding site that overlaps the U11 site, and the interaction with the 3′ splice site correlated with U1, not U11, binding. These results show that the NRS can interact with a 3′ splice site and suggest that U1 is of primary importance for NRS splicing inhibition.

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 Human Autoimmune Sera as Molecular Probes for the Identification of an Autoantigen Kinase Signaling Pathway

2002 ◽  
Vol 196 (9) ◽  
pp. 1213-1226 ◽  
Author(s):  
Makoto Kamachi ◽  
Truc M. Le ◽  
Susan J. Kim ◽  
Meghan E. Geiger ◽  
Paul Anderson ◽  
...  
Keyword(s):  
Topoisomerase I ◽  
Molecular Probes ◽  
Splicing Factors ◽  
Apoptotic Cells ◽  
Sr Protein ◽  
Small Nuclear Ribonucleoprotein ◽  
Alternative Mrna Splicing ◽  
U1 Snrnp

Using human autoimmune sera as molecular probes, we previously described the association of phosphorylated serine/arginine splicing factors (SR splicing factors) with the U1-small nuclear ribonucleoprotein (U1-snRNP) and U3-small nucleolar RNP (snoRNP) in apoptotic cells. SR proteins are highly conserved autoantigens whose activity is tightly regulated by reversible phosphorylation of serine residues by at least eight different SR protein kinase kinases (SRPKs), including SRPK1, SRPK2, and the scleroderma autoantigen topoisomerase I. In this report, we demonstrate that only one of the known SRPKs, SRPK1, is associated with the U1-snRNP autoantigen complex in healthy and apoptotic cells. SRPK1 is activated early during apoptosis, followed by caspase-mediated proteolytic inactivation at later time points. SRPKs are cleaved in vivo after multiple apoptotic stimuli, and cleavage can be inhibited by overexpression of bcl-2 and bcl-xL, and by exposure to soluble peptide caspase inhibitors. Incubation of recombinant caspases with in vitro–translated SRPKs demonstrates that SRPK1 and SRPK2 are in vitro substrates for caspases-8 and -9, respectively. In contrast, topoisomerase I is cleaved by downstream caspases (-3 and -6). Since each of these SRPKs sits at a distinct checkpoint in the caspase cascade, SRPKs may serve an important role in signaling pathways governing apoptosis, alternative mRNA splicing, SR protein trafficking, RNA stability, and possibly the generation of autoantibodies directed against splicing factors.

scholarly journals Elongin A regulates transcription in vivo through enhanced RNA polymerase processivity

2020 ◽  
pp. jbc.RA120.015876
Author(s):  
Yating Wang ◽  
Liming Hou ◽  
M. Behfar Ardehali ◽  
Robert E. Kingston ◽  
Brian D Dynlacht
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Elongation ◽  
Rna Seq ◽  
Transcription Profiles ◽  
Genome Wide ◽  
The Impact

Elongin is an RNA polymerase II (RNAPII)-associated factor that has been shown to stimulate transcriptional elongation in vitro. The Elongin complex is thought to be required for transcriptional induction in response to cellular stimuli and to ubiquitinate RNAPII in response to DNA damage. Yet the impact of the Elongin complex on transcription in vivo has not been well studied. Here, we performed comprehensive studies of the role of Elongin A, the largest subunit of the Elongin complex, on RNAPII transcription genome-wide. Our results suggest that Elongin A localizes to actively transcribed regions and potential enhancers, and the level of recruitment correlated with transcription levels. We also identified a large group of factors involved in transcription as Elongin A-associated factors. In addition, we found that loss of Elongin A leads to dramatically reduced levels of Ser2-phosphorylated, but not total, RNAPII, and cells depleted of Elongin A show stronger promoter RNAPII pausing, suggesting that Elongin A may be involved in the release of paused RNAPII. Our RNA-seq studies suggest that loss of Elongin A did not alter global transcription, and unlike prior in vitro studies, we did not observe a dramatic impact on RNAPII elongation rates in our cell-based nascent RNA-seq experiments upon Elongin A depletion. Taken together, our studies provide the first comprehensive analysis of the role of Elongin A in regulating transcription in vivo. Our studies also revealed that unlike prior in vitro findings, depletion of Elongin A has little impact on global transcription profiles and transcription elongation in vivo.

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