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,Benjamin J Blencowe

doi:10.1074/jbc.m507410200

Abstract

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 SMC1alpha, 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.

Highlights

The development of high throughput procedures for gel-free tandem mass spectrometry-based identification of factors in multisubunit complexes has provided a wealth of information on the functional associations of proteins [1,2,3]
The characterization by tandem mass spectrometry of immunopurified SRm160/300-containing complexes in this study resulted in the discovery of new factors that interact with these proteins
The detection of associations with many new factors involved in pre-mRNA processing, as well as the identification of associated factors that function in chromatin remodeling, suggests that SRm160, possibly in association with SRm300, could function in the coupling of pre-mRNA processing with transcription via interactions that impact on chromatin

Summary

EXPERIMENTAL PROCEDURES

Immunopurification of Endogenous Complexes—Prior to immunoprecipitation, nuclear extract was preincubated for 15 min at 30 °C under splicing conditions (2 mM MgCl2, 1.5 mM ATP, and 5 mM phosphocreatine) with the addition of an RNase mixture (16 ng/␮l; Roche Applied Science) and DNase I (0.3 units/␮l) and in the presence of phosphatase inhibitors (1 mM potassium fluoride, 0.1 mM sodium pyrophosphate, and 1 mM sodium ␤-glycerophosphate). The beads were washed three times (1.5 ml) with 100 mM NaCl, 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 0.1% Nonidet P-40, and 1 mM dithiothreitol and eluted with 2 M NaCl, 10 mM HEPES (pH 7.5), and 1 mM EDTA (200 ␮l), followed by a brief wash with 10 mM HEPES (pH 7.5) and 1 mM EDTA (200 ␮l) These pooled eluates were back-bound with protein A-Sepharose and with rabbit anti-mouse IgG/IgM-coated protein A-Sepharose for 30 min at 4 °C with rotation. Gel Filtration—HeLa nuclear extract (10 mg) was incubated with ATP, Mg2ϩ, and phosphocreatine as well as RNase and DNase and loaded onto a 1.5 ϫ 28-cm Sephacryl S400 column equilibrated with 20 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM MgCl2, 1 mM dithiothreitol, 10% glycerol, 1 mM ␤-glycerophosphate, 1 mM KF, and 0.1 mM sodium pyrophosphate. Nuclear spreads for immunofluorescence studies were performed as described [26]

RESULTS AND DISCUSSION

TABLE ONE

Scaffold attachment factor B

RNAi phenotype