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J. Biol. Chem., Vol. 280, Issue 51, 42227-42236, December 23, 2005

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Proteomic Analysis of SRm160-containing Complexes Reveals a Conserved Association with Cohesin^*

Susan McCracken, Dasa Longman, Edyta Marcon¶, Peter Moens¶, Michael Downey||, Jeffrey A. Nickerson**, Rolf Jessberger, Andrew Wilde||, Javier F. Caceres, Andrew Emili||, and Benjamin J. Blencowe||1

From the Banting and Best Department of Medical Research, C. H. Best Institute, and the ^||Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5G 1L6, Canada, the Medical Research Council Human Genetics Unit, Edinburgh EH4 2XU, Scotland, United Kingdom, the ^¶Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada, the ^**Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, and the Institute of Physiological Chemistry, Medical School, Medizinisch-Theoretisches Zentrum Technical University, Fiedlerstrasse 42, D-01307 Dresden, Germany

In this study, we describe a rapid immunoaffinity purification procedure for gel-free tandem mass spectrometry-based analysis of endogenous protein complexes and apply it to the characterization of complexes containing the SRm160 (serine/arginine repeat-related nuclear matrix protein of 160 kDa) splicing coactivator. In addition to promoting splicing, SRm160 stimulates 3'-end processing via its N-terminal PWI nucleic acid-binding domain and is found in a post-splicing exon junction complex that has been implicated in coupling splicing with mRNA turnover, export, and translation. Consistent with these known functional associations, we found that the majority of proteins identified in SRm160-containing complexes are associated with pre-mRNA processing. Interestingly, SRm160 is also associated with factors involved in chromatin regulation and sister chromatid cohesion, specifically the cohesin subunits SMC1, SMC3, RAD21, and SA2. Gradient fractionation suggested that there are two predominant SRm160-containing complexes, one enriched in splicing components and the other enriched in cohesin subunits. Co-immunoprecipitation and co-localization experiments, as well as combinatorial RNA interference in Caenorhabditis elegans, support the existence of conserved and functional interactions between SRm160 and cohesin.

Received for publication, July 8, 2005 , and in revised form, September 8, 2005.

^* This work was supported by operating grants from the Canadian Institutes of Health Research and the National Cancer Institute of Canada (to B. J. B.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1 and 2.

¹ To whom correspondence should be addressed: Banting and Best Dept. of Medical Research, C. H. Best Inst., Rm. 410, University of Toronto, 112 College St., Toronto, Ontario M5G 1L6, Canada. Tel.: 416-978-3016; Fax: 416-978-8528; E-mail: b.blencowe{at}utoronto.ca.

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