Moens P, Downey M, Longman D, McCracken S, Blencowe BJ, Jessberger R, Marcon E, Wilde A, Nickerson JA, Emili A, Caceres JF
[
J Biol Chem,
2005]
In this report 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 160kDa) 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 to mRNA turnover, export and translation. Consistent with these known functional associations, we find 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, SMC1alpha, SMC3, hRAD21 and SA2. Gradient fractionation suggests that there are two predominant SRm160-containing complexes, one enriched in splicing components and the other enriched in cohesin subunits. Co-immunoprecipitation and colocalization experiments, as well as combinatorial RNA interference in C. elegans, support the existence of conserved and functional interactions between SRm160 and cohesin.