RNA Polymerase II Targets Pre-mRNA Splicing Factors to Transcription Sites In Vivo

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.

Download Full-text

Distribution of pre-mRNA splicing factors at sites of RNA polymerase II transcription.

Genes & Development ◽

10.1101/gad.11.9.1148 ◽

1997 ◽

Vol 11 (9) ◽

pp. 1148-1159 ◽

Cited By ~ 88

Author(s):

K M Neugebauer ◽

M B Roth

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Mrna Splicing ◽

Splicing Factors ◽

Rna Polymerase Ii Transcription

Download Full-text

RNA Polymerase II Carboxy-Terminal Domain Phosphorylation Is Required for Cotranscriptional Pre-mRNA Splicing and 3′-End Formation

Molecular and Cellular Biology ◽

10.1128/mcb.24.20.8963-8969.2004 ◽

2004 ◽

Vol 24 (20) ◽

pp. 8963-8969 ◽

Cited By ~ 78

Author(s):

Gregory Bird ◽

Diego A. R. Zorio ◽

David L. Bentley

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Kinase Inhibitors ◽

Mrna Splicing ◽

Pol Ii ◽

Terminal Domain ◽

Carboxy Terminal Domain ◽

Carboxy Terminal ◽

Ctd Phosphorylation

ABSTRACT We investigated the role of RNA polymerase II (pol II) carboxy-terminal domain (CTD) phosphorylation in pre-mRNA processing coupled and uncoupled from transcription in Xenopus oocytes. Inhibition of CTD phosphorylation by the kinase inhibitors 5,6-dichloro-1β-d-ribofuranosyl-benzimidazole and H8 blocked transcription-coupled splicing and poly(A) site cleavage. These experiments suggest that pol II CTD phosphorylation is required for efficient pre-mRNA splicing and 3′-end formation in vivo. In contrast, processing of injected pre-mRNA was unaffected by either kinase inhibitors or α-amanitin-induced depletion of pol II. pol II therefore does not appear to participate directly in posttranscriptional processing, at least in frog oocytes. Together these experiments show that the influence of the phosphorylated CTD on pre-mRNA splicing and 3′-end processing is mediated by transcriptional coupling.

Download Full-text

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

The Journal of Cell Biology ◽

10.1083/jcb.143.2.297 ◽

1998 ◽

Vol 143 (2) ◽

pp. 297-307 ◽

Cited By ~ 184

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.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.24.20.9176-9185.2004 ◽

2004 ◽

Vol 24 (20) ◽

pp. 9176-9185 ◽

Cited By ~ 60

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.

Download Full-text

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

Molecular Biology of the Cell ◽

10.1091/mbc.7.10.1559 ◽

1996 ◽

Vol 7 (10) ◽

pp. 1559-1572 ◽

Cited By ~ 109

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.

Download Full-text

Distribution of B52 within a chromosomal locus depends on the level of transcription.

Molecular Biology of the Cell ◽

10.1091/mbc.5.1.71 ◽

1994 ◽

Vol 5 (1) ◽

pp. 71-79 ◽

Cited By ~ 11

Author(s):

D T Champlin ◽

J T Lis

Keyword(s):

Heat Shock ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Polytene Chromosomes ◽

Mrna Splicing ◽

Chromosomal Locus ◽

Highly Active ◽

Heat Shocks

Drosophila B52 protein is a homologue of human ASF/SF2 that functions in vitro as an essential pre-mRNA splicing factor. Immunofluorescence analysis of polytene chromosomes has shown that B52 generally colocalizes with RNA polymerase II; however, in contrast to other splicing factors, B52 brackets RNA polymerase II at highly active heat-shock puffs. Also, UV cross-linking in nonpolytene cells has shown that B52 cross-links in vivo to DNA flanking the highly active transcription units. Here, we find that the distribution of cross-linked B52 at heat-shock loci depends on transcription levels. Heat shocks at low and moderate temperatures, which induce corresponding levels of transcription, recruit B52 both to transcribed DNA and to flanking DNA, whereas a full heat-shock induction concentrates B52 on the DNA that brackets the entire activated region. We have also identified a 46-kDa protein from Chironomus tentans that binds Drosophila B52 antibodies and has a distribution on chromosomes analogous to B52. This protein is found throughout the moderately transcribed Balbiani rings. However, when transcription at these rings is hyperinduced to levels comparable to fully induced Drosophila heat-shock genes, the protein is restricted to the boundaries of highly decondensed chromatin. We suggest that B52 tracks to chromatin fibers that are folding or unfolding, and we discuss this in light of B52's proposed roles in pre-mRNA splicing and control.

Download Full-text

Defective histone supply causes changes in RNA polymerase II elongation rate and cotranscriptional pre-mRNA splicing

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1506760112 ◽

2015 ◽

Vol 112 (48) ◽

pp. 14840-14845 ◽

Cited By ~ 32

Author(s):

Silvia Jimeno-González ◽

Laura Payán-Bravo ◽

Ana M. Muñoz-Cabello ◽

Macarena Guijo ◽

Gabriel Gutierrez ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Chromatin Structure ◽

Intron Retention ◽

Direct Consequence ◽

Elongation Rate ◽

Mrna Splicing ◽

Mrna Levels

RNA polymerase II (RNAPII) transcription elongation is a highly regulated process that greatly influences mRNA levels as well as pre-mRNA splicing. Despite many studies in vitro, how chromatin modulates RNAPII elongation in vivo is still unclear. Here, we show that a decrease in the level of available canonical histones leads to more accessible chromatin with decreased levels of canonical histones and variants H2A.X and H2A.Z and increased levels of H3.3. With this altered chromatin structure, the RNAPII elongation rate increases, and the kinetics of pre-mRNA splicing is delayed with respect to RNAPII elongation. Consistent with the kinetic model of cotranscriptional splicing, the rapid RNAPII elongation induced by histone depletion promotes the skipping of variable exons in the CD44 gene. Indeed, a slowly elongating mutant of RNAPII was able to rescue this defect, indicating that the defective splicing induced by histone depletion is a direct consequence of the increased elongation rate. In addition, genome-wide analysis evidenced that histone reduction promotes widespread alterations in pre-mRNA processing, including intron retention and changes in alternative splicing. Our data demonstrate that pre-mRNA splicing may be regulated by chromatin structure through the modulation of the RNAPII elongation rate.

Download Full-text

Splicing components are excluded from the transcriptionally inactive XY body in male meiotic nuclei.

Molecular Biology of the Cell ◽

10.1091/mbc.5.12.1341 ◽

1994 ◽

Vol 5 (12) ◽

pp. 1341-1352 ◽

Cited By ~ 26

Author(s):

C Richler ◽

G Ast ◽

R Goitein ◽

J Wahrman ◽

R Sperling ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Indirect Immunofluorescence ◽

Mrna Splicing ◽

Splicing Factors ◽

Gradual Reduction ◽

Xy Body ◽

Nuclear Domain ◽

Distribution Of Elements ◽

Splicing Machinery

The study of the effect of programmed cessation of transcription in a large nuclear domain, on the distribution of elements of the pre-mRNA splicing machinery, is the main aim of this paper. To this end, we took advantage of the nuclear partitioning of mouse spermatocytes early in meiosis into autosomal transcribing and XY nontranscribing compartments. This system also allows to extend this study to stages in sperm differentiation that are accompanied by reduction and eventual cessation of transcription. We show by indirect immunofluorescence in spermatogenetic cells that 1) fluorescent signals of the pre-mRNA splicing factors SF53/4 and SC35, of the Sm antigens, and of RNA polymerase II, are largely absent from the nontranscribing, X-inactivated compartment, but are abundantly present in the transcribing autosomal compartment and 2) the presence, gradual reduction, and absence of transcriptive activity in nuclei undergoing the sperm formation sequence are positively correlated with the fluorescence patterns of the antibodies against SF53/4, SC35, and the Sm antigens. These data suggest that cessation of transcription during spermatogenesis is accompanied by exclusion of the splicing machinery from nontranscribing chromatin to its vicinity.

Download Full-text