The polypyrimidine tract binding protein (PTB) represses splicing of exon 6B from the beta-tropomyosin pre-mRNA by directly interfering with the binding of the U2AF65 subunit.
Splicing of exon 6B from the beta-tropomyosin pre-mRNA is repressed in nonmuscle cells and myoblasts by a complex array of intronic elements surrounding the exon. In this study, we analyzed the proteins that mediate splicing repression of exon 6B through binding to the upstream element. We identified the polypyrimidine tract binding protein (PTB) as a component of complexes isolated from myoblasts that assemble onto the branch point region and the pyrimidine tract. In vitro splicing assays and PTB knockdown experiments by RNA interference demonstrated that PTB acts as a repressor of splicing of exon 6B. Using psoralen experiments, we showed that PTB acts at an early stage of spliceosome assembly by preventing the binding of U2 snRNA on the branch point. Using UV cross-linking and immunoprecipitation experiments with site-specific labeled RNA in PTB-depleted nuclear extracts, we found that the decrease in PTB was correlated with an increase in U2AF65. In addition, competition experiments showed that PTB is able to displace the binding of U2AF65 on the polypyrimidine tract. Our results strongly support a model whereby PTB competes with U2AF65 for binding to the polypyrimidine tract.
Saulière
Jérôme
J
Centre de Génétique Moléculaire, UPR2167, CNRS, 1 avenue de la Terrasse, 91198 Gif sur Yvette, France.
Sureau
Alain
A
Expert-Bezançon
Alain
A
Marie
Joëlle
J
D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D005091:Exons; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D032446:Myoblasts / Q000166:cytology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010449:Peptide Mapping; D038941:Polypyrimidine Tract-Binding Protein / Q000235:genetics / Q000378:metabolism*; D011784:Quail; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D016622:Silver Staining; D000072668:Splicing Factor U2AF; D014162:Transfection; D014335:Tropomyosin / Q000235:genetics*
D009687:Nuclear Proteins; D012322:RNA Precursors; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; D014335:Tropomyosin; C000607494:U2AF2 protein, human; D038941:Polypyrimidine Tract-Binding Protein; D005982:Glutathione Transferase