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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.
Mol Cell Biol. 2006 Dec; 26(23):8755-69.MC

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.

Authors+Show Affiliations

Centre de Génétique Moléculaire, UPR2167, CNRS, 1 avenue de la Terrasse, 91198 Gif sur Yvette, France.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16982681

Citation

Saulière, Jérôme, et al. "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, pp. 8755-69.
Saulière J, Sureau A, Expert-Bezançon A, et al. 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. Mol Cell Biol. 2006;26(23):8755-69.
Saulière, J., Sureau, A., Expert-Bezançon, A., & Marie, J. (2006). 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, 26(23), 8755-69.
Saulière J, et al. 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. Mol Cell Biol. 2006;26(23):8755-69. PubMed PMID: 16982681.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Saulière,Jérôme, AU - Sureau,Alain, AU - Expert-Bezançon,Alain, AU - Marie,Joëlle, Y1 - 2006/09/18/ PY - 2006/9/20/pubmed PY - 2006/12/27/medline PY - 2006/9/20/entrez SP - 8755 EP - 69 JF - Molecular and cellular biology JO - Mol Cell Biol VL - 26 IS - 23 N2 - 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. SN - 0270-7306 UR - https://www.unboundmedicine.com/medline/citation/16982681/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_ L2 - http://mcb.asm.org/cgi/pmidlookup?view=long&pmid=16982681 DB - PRIME DP - Unbound Medicine ER -