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J. Biol. Chem., Vol. 278, Issue 50, 50031-50039, December 12, 2003

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Reprogramming Alternative Pre-messenger RNA Splicing through the Use of Protein-binding Antisense Oligonucleotides^*

Jonathan Villemaire, Isabelle Dion, Sherif Abou Elela, and Benoit Chabot¶

From the Département de microbiologie et d'infectiologie, RNA/RNP Group, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada

Alternative pre-messenger RNA splicing is a major contributor to proteomic diversity in higher eukaryotes and represents a key step in the control of protein function in a large variety of biological systems. As a means of artificially altering splice site choice, we have investigated the impact of positioning proteins in the vicinity of 5' splice sites. We find that a recombinant GST-MS2 protein interferes with 5' splice site use, most efficiently when it binds upstream of that site. To broaden the use of proteins as steric inhibitors of splicing, we have tested the activity of antisense oligonucleotides carrying binding sites for the heterogeneous nuclear ribonucleoprotein A1/A2 proteins. In a HeLa cell extract, tailed oligonucleotides complementary to exonic sequences elicit strong shifts in 5' splice site selection. In four different human cell lines, an interfering oligonucleotide carrying A1/A2 binding sites also shifted the alternative splicing of the Bcl-x pre-mRNA more efficiently than oligonucleotides acting through duplex formation only. The use of protein-binding oligonucleotides that interfere with U1 small nuclear ribonucleoprotein binding therefore represents a novel and powerful approach to control splice site selection in cells.

Received for publication, August 12, 2003 , and in revised form, September 25, 2003.

^* This work was supported in part by a grant from the National Cancer Institute of Canada with funds from the Canadian Cancer Society, and Genome Québec and Genome Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a studentship from the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche (FCAR).

Research Scholar Junior II from the Fonds de la Recherche en Santé du Québec (FRSQ).

^¶ Canada Research Chair in Functional Genomics. To whom correspondence should be addressed: Département de microbiologie et d'infectiologie, Faculté de Médecine, Université de Sherbrooke, 3001, 12e avenue Nord, Sherbrooke, Québec J1H 5N4, Canada. Tel.: 819-564-5295; Fax: 819-564-5392; E-mail: Benoit.Chabot{at}USherbrooke.ca.

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