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PMID: 16982681 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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.

Molecular and cellular biology ·Vol. 26 ·No. 23 ·2006-12-00 ·Pages 8755-69

Saulière J, Sureau A, Expert-Bezançon A, Marie J

Abstract

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.

MeSH Terms
Animals Cell Nucleus/chemistry,metabolism Cells, Cultured Exons Glutathione Transferase/metabolism HeLa Cells Humans Myoblasts/cytology Nuclear Proteins/genetics,metabolism Peptide Mapping Polypyrimidine Tract-Binding Protein/genetics,metabolism Quail RNA Precursors/genetics,metabolism RNA Splicing RNA, Small Interfering/metabolism Recombinant Proteins/isolation & purification,metabolism Ribonucleoproteins/genetics,metabolism Silver Staining Splicing Factor U2AF Transfection Tropomyosin/genetics
Chemicals
Nuclear Proteins RNA Precursors RNA, Small Interfering Recombinant Proteins Ribonucleoproteins Splicing Factor U2AF Tropomyosin U2AF2 protein, human Polypyrimidine Tract-Binding Protein Glutathione Transferase
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Saulière Jérôme
Centre de Génétique Moléculaire, UPR2167, CNRS, 1 avenue de la Terrasse, 91198 Gif sur Yvette, France.
Sureau Alain
Expert-Bezançon Alain
Marie Joëlle
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.00893-06
Published
2006-12-00
Epub
2006-00-18
Pages
8755-69
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC1636812
Subset
IM
External Links
PubMed source DOI: 10.1128/MCB.00893-06 PMC Full Text View journal details
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