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Originally published In Press as doi:10.1074/jbc.M507410200 on September 13, 2005

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*{boxs}

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

From the {ddagger}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 {ddagger}{ddagger}Institute of Physiological Chemistry, Medical School, Medizinisch-Theoretisches Zentrum Technical University, Fiedlerstrasse 42, D-01307 Dresden, Germany

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
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{alpha}, 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
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 (1–3). Often, proteins targeted for purification are expressed with affinity tags, allowing coupling to affinity resins with antibodies or ligands specific for the tag moieties. Unlike the situation in yeast, mammalian systems are not amenable to efficient genetic recombination procedures allowing the expression of epitope-tagged proteins under the control of endogenous promoters. Consequently, many affinity purification procedures have been performed using tagged factors overexpressed by transient transfection or from stably integrated constructs. Overexpression often has the major drawback of resulting in nonspecific associations with the tagged factor, and developing tagged cell lines is time-consuming and laborious.

An alternative to the purification of tagged, exogenously expressed proteins is to target endogenous proteins with specific antibodies. This affords the main advantage of allowing the recovery of proteins associated with endogenous levels of the targeted proteins, thereby avoiding overexpression artifacts. In this study, we utilized this approach to investigate the composition of complexes associated with SRm160, a splicing coactivator that is also implicated in the coupling of different steps in gene expression at the post-transcriptional level (4–8).

SRm160 was originally identified as a nuclear matrix antigen that is highly concentrated in interphase nuclear "speckle" domains (interchromatin granule clusters) enriched in splicing components (9, 10). Interestingly, unlike other splicing components concentrated in speckles, SRm160 undergoes a dramatic redistribution during mitosis, becoming concentrated at the spindle poles and at the spindle (4, 10). Subsequent cloning of an SRm160 cDNA revealed that it contains an arginine/serine repeat domain, a feature of many defined splicing factors (4). Biochemical experiments showed that SRm160 (i) associates with splicing complexes, (ii) functions as a coactivator of constitutive and exon enhancer-dependent splicing, and (iii) stimulates 3'-end formation (4, 5, 7, 8, 11). SRm160 remains bound to processed mRNA in an exon junction complex located ~20–24 nucleotides upstream of exon-exon junctions (6, 12, 13). Although the precise role of SRm160 in the exon junction complex is not known, other factors in this complex have been implicated as adaptors that link to mRNA export, turnover by nonsense-mediated mRNA decay, and translation (14).

To gain further insight into the cellular functions of SRm160, complexes affinity-purified with different SRm160-specific antibodies were analyzed for novel and conserved interactions. In addition to generating an extensive data base of associated pre-mRNA processing components, factors associated with transcription, chromatin regulation, and sister chromatid cohesion were identified in the purified complexes. Interestingly, a complete set of subunits associated with the mitotic cohesin complex co-purified with SRm160, including SMC1{alpha} (structural maintenance of chromosome 1{alpha}), SMC3, human (h)2 RAD21 (double-strand break repair protein rad21 homolog), and SA2 (stromal antigen 2).

Cohesin is essential for sister chromatid cohesion, ensuring correct chromosome segregation (15, 16). In higher eukaryotes, it is composed of four subunits, a subset of which corresponds to mitosis- and meiosis-specific isotypes. The subunits hRAD21 and SA1/2 function in mitosis, whereas SMC1{beta}, REC8, and STAG3 function during meiosis, and both SMC1{alpha} and SMC3 function in mitosis and meiosis (reviewed in Refs. 17 and 18). Subunits of the mitotic cohesin complex are also important for DNA repair and the ATM-dependent S phase checkpoint (reviewed in Ref. 18).

The interaction between SRm160 and cohesin subunits identified in this study is conserved in Xenopus laevis. We also found that simultaneous knockdown by RNA interference (RNAi) of Caenorhabditis elegans (Ce) SRm160 and coh-1 (an ortholog of rad21) results in an early embryonic lethal phenotype, whereas single RNAi of coh-1 results in an uncoordinated phenotype, and single RNAi of SRm160 results in no apparent phenotype. This suggests that SRm160 and coh-1 interact genetically in the same pathway. Moreover, consistent with a possible cellular function of SRm160 in sister chromatid cohesion or a cohesin-related activity, SRm160 localizes to the cohesin-containing core of synaptonemal complexes in spermatocytes, at the pachytene stage of meiotic prophase I. Together, our results provide evidence for a conserved functional association between SRm160 and cohesins.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
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/µl; Roche Applied Science) and DNase I (0.3 units/µl) and in the presence of phosphatase inhibitors (1 mM potassium fluoride, 0.1 mM sodium pyrophosphate, and 1 mM sodium {beta}-glycerophosphate). A similar treatment was used prior to loading the sample onto a gel filtration column. Protein A-Sepharose (30 µl of packed beads) was loaded with rabbit anti-mouse IgG and IgM antibody (72 µg), monoclonal antibody (mAb) 8WG16 (50 µg), antigen affinity-purified rabbit anti-SRm160 polyclonal antibody (pAb) (50 µg), or rabbit anti-SRm300 serum (25 µl), followed by cross-linking with 20 mM dimethylpimelimidate (19) and loading of the control IgM protein (Sigma) or mAb B1C8 (1.5 ml of culture supernatant, ~150 µg) onto rabbit anti-mouse antibody-coated beads. Control immunoprecipitations were performed with rabbit anti-mouse antibody-coated beads with or without the control IgM protein. HeLa nuclear extract (1.5 mg) (20) or cytostatic factor-arrested egg extract from X. laevis (1.5 mg) (21) was preincubated with ATP, Mg2+, and phosphocreatine as well as DNase and RNase (7) at 30 °C for 15 min. The extract was incubated with the beads for 3 h at 4 °C with gentle rotation in 60 mM NaCl, 13 mM HEPES (pH 7.9), 1.4 mM MgCl2, 14% glycerol, 0.5 mM dithiothreitol, 0.7 mM {beta}-glycerophosphate, 0.7 mM NaF, and 0.07 mM sodium pyrophosphate in a final volume of 750 µl. 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 µl), followed by a brief wash with 10 mM HEPES (pH 7.5) and 1 mM EDTA (200 µl). 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. After removal of the beads, the samples were bound to 10 µl of phenyl-Sepharose for 1 h at 4 °C and then collected by centrifugation. The beads were eluted with 50 µlof8 M urea and 10 mM Tris (pH 8.8), followed by a wash with 50 µlof 10 mM Tris (pH 8.8). The pooled eluates were then digested with trypsin prior to mass spectrometry, which was performed as described (22). If the immunoprecipitates were to be analyzed by Western blotting, the phenyl-Sepharose step was omitted, and the samples were precipitated with 20% trichloroacetic acid and 1.5 mg/ml sodium deoxycholate, washed with 10% trichloroacetic acid and with acetone, resuspended in SDS sample buffer, and analyzed by SDS-PAGE.

Antisera—Analysis of SRm160-containing fractions was performed using mAb B1C8 (mouse) (10), rabbit anti-SRm160 pAb (4), and goat anti-SRm160 K15 pAb (a commercial affinity-purified peptide-specific antibody raised against the SRm160 C terminus; Santa Cruz Biotechnology, Inc.). Rabbit anti-SRm300, anti-SMC1{alpha}, and anti-SMC3 pAbs have been described previously (4, 23). Rabbit anti-hRAD21 and anti-transcription factor IIF pAbs, mAb 104, and mAb Y12 were generous gifts from Jan Michael Peters, Jack Greenblatt, Mark Roth, and Joan Steitz, respectively.

Synchronization of Cells—Cells were synchronized by double thymidine block using two cycles of block with 2 mM thymidine for 16 h, followed by release. G1/S phase cells were harvested directly from the blocked cells; mitotic cells were released from the block and incubated in fresh medium until entry into mitosis.

Immunoblotting—Proteins were transferred from SDS-polyacrylamide gels by wet electrotransfer overnight, and immunoblot analysis was performed as described previously (7).

Gel Filtration—HeLa nuclear extract (10 mg) was incubated with ATP, Mg2+, and phosphocreatine as well as RNase and DNase and loaded onto a 1.5 x 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 {beta}-glycerophosphate, 1 mM KF, and 0.1 mM sodium pyrophosphate. Fractions were collected and analyzed by immunoblotting.

RNAi in C. elegans—RNAi in C. elegans was performed as described previously (24).

Immunofluorescence—Mouse spermatocytes for immunoblot analysis were prepared as described (25). Nuclear spreads for immunofluorescence studies were performed as described (26).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Proteomic Analysis of SRm160-containing Complexes—A rapid immunoaffinity purification procedure was developed for the gel-free mass spectrometric analysis of SRm160-containing complexes in HeLa nuclear extract. This procedure, which is potentially applicable to any endogenous protein complex for which a specific and immunoprecipitation-competent antibody exists, is outlined in Fig. 1A and summarized in the legend. A more detailed description is also given under "Experimental Procedures."

Two different antibodies that are highly specific for SRm160 were employed in multiple independent immunoaffinity purifications. One of these antibodies, mAb B1C8 (10), is a murine monoclonal antibody that was originally used to purify SRm160 (4). The other antibody (rabbit anti-SRm160 pAb) is an antigen affinity-purified rabbit polyclonal antibody raised against the conserved N-terminal domain of SRm160 expressed as a glutathione S-transferase fusion protein. The specificity of these antibodies for SRm160 has been demonstrated in several ways. In immunoblots of total nuclear extract, both antibodies selectively recognize an ~160-kDa antigen that has been identified as SRm160 by fractionation and peptide microsequencing (4, 27). In addition, both antisera specifically recognized baculovirus-expressed recombinant SRm160,3 and both have very similar properties in different bioassays. For example, both antibodies preferentially immunoprecipitate exon-containing splicing complexes from HeLa nuclear extract, and both specifically stain speckles in interphase nuclei and the spindle apparatus at mitosis (4). In addition, depletion of SRm160 from HeLa nuclear extract using rabbit anti-SRm160 pAb prevents pre-mRNA splicing, and activity can be restored to the depleted extract by addition of purified recombinant protein (28).



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  		FIGURE 1.
Co-immunoprecipitation of SRm160 and subunits of the cohesin complex. A: the procedure for immunopurification of SRm160-containing complexes is outlined. HeLa nuclear extract was first pretreated extensively with RNase and DNase to remove any endogenous nucleic acids (7). The pretreated extract was incubated with protein A-Sepharose beads covalently coupled to anti-SRm160 antibody. After wash steps, bound proteins were eluted in high salt. The high salt eluate was then back-cleared to remove immunoglobulin chains released from the protein A beads. The remaining proteins were concentrated by hydrophobic binding to phenyl-Sepharose, eluted in 8 M urea, and subjected to trypsin digestion in solution, and the peptide mixtures were analyzed by capillary-scale shotgun liquid chromatography-tandem mass spectrometry (1). IP, immunoprecipitation. B: the factors in immunoprecipitated SRm160/300-containing complexes were detected. Samples were immunoprecipitated with control beads (lane 2), mAb B1C8 (lane 3), rabbit anti-SRm300 pAb (rAb {alpha}SRm300; lane 4), or mAb 8WG16 (lane 5); analyzed by SDS-PAGE; and immunoblotted with the antibodies indicated to the right. The load sample (lane 1) contained 4% of the amount of input used in the immunoprecipitations. TFIIF, transcription factor IIF. C: SRm160 was immunoprecipitated with 25 µl of rabbit anti-SRm160 pAb (lane 4) or with 25, 50, 100, or 200 µl of mAb B1C8 culture supernatant (lanes 5–8) and immunoblotted with the antibodies indicated to the right. Loads 1 and 2 correspond to 1.6 and 3.3% of the input to the immunoprecipitation (lanes 1 and 2, respectively). D: lower panel, G1/S phase (lanes 2 and 4) and mitotic (lanes 3 and 5) extracts of HeLa cells were prepared from synchronized cells. Synchronization was monitored by analysis for Tyr15 phosphorylation of Cdc2. Upper panel, control antibody and mAb B1C8 immunoprecipitates from these and asynchronous extracts (lanes 4–9) were analyzed for SMC1 and hRAD21 by immunoblotting. The loads (lanes 1–3) represent 7% of the input used for each immunoprecipitation. HeLa NE, HeLa nuclear extract.

 
Also analyzed by mass spectrometry using our rapid immunopurification procedure were complexes affinity-purified with a rabbit polyclonal antibody specific for SRm300 (serine/arginine repeat-related nuclear matrix protein of 300 kDa) (rabbit anti-SRm300 pAb) (28), and complexes were immunopurified with a murine monoclonal antibody specific for the hypophosphorylated form of the largest subunit of RNA polymerase II (pol II) (mAb 8WG16) (29). Rabbit anti-SRm300 pAb was raised against a glutathione S-transferase fusion protein containing a conserved N-terminal domain of SRm300 and has been shown to specifically recognize SRm300 in HeLa nuclear extract, to immunolabel speckles, and to immunoprecipitate exon-containing splicing complexes (28). Based on previous results demonstrating that the majority of SRm300 in HeLa nuclear extracts is tightly associated with SRm160, whereas a subpopulation of SRm160 exists that is not bound to SRm300 (4, 27, 28), we expected to detect a subset of SRm160-associated proteins in the SRm300-containing complexes. In contrast, previous co-immunoprecipitation analyses and fractionation studies did not reveal any evidence for an association between SRm160-containing and pol II-containing complexes (30, 31); and therefore, we did not expect to observe a significant number of proteins that are shared between these complexes.

Consistent with our previous results (4, 27, 28), 36% of the proteins detected with high confidence (see below) in complexes immunopurified with rabbit anti-SRm300 pAb overlapped with proteins in the immunopurified SRm160-containing complexes (TABLE ONE). In contrast, the profile of proteins detected in the immunoaffinity purification using mAb 8WG16 was quite distinct from the profiles observed for the SRm160/300 purifications (supplemental Table 1). Finally, no proteins other than the trypsinogen precursor were detected by mass spectrometry in samples recovered from beads alone or from beads coupled to a control antibody (rabbit anti-Ig; data not shown). These results indicate that our immunoaffinity purification procedure results in the efficient and specific recovery of proteins associated with immunoaffinity-purified complexes.


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  		TABLE ONE
Mass spectrometric identification of polypeptides recovered from complexes immunopurified with anti-SRm160 antibodies (mAb B1C8 and rabbit anti-SRm160pAb) and anti-SRm300 pAb The number of independently detected peptides is indicated for each immunopurification. Only proteins identified by three or more independent and unambiguous spectra for separate hits with any one antibody are listed. Proteins that co-immunoprecipitated with control beads or with mAb 8WG16 (anti-pol II) were excluded from the list, regardless of their detection of the SRm160/300-containing complexes. hnRNP, heterogeneous nuclear ribonucleoprotein; eIF, eukaryotic initiation factor.

 
Supplemental Table 1 includes a complete list of proteins identified by mass spectrometry in multiple independent purifications with each antibody, along with information on the number of peptide spectra detected for each protein. The large number of peptides identified in these analyses reflects the sensitivity of the gel-free tandem mass spectrometric method. Proteins considered to associate with SRm160 with relatively high specificity and affinity are shown in TABLE ONE; these were detected by three or more independent peptide spectra in one or more purifications with either of the two anti-SRm160 antibodies and were not detected by mass spectrometry in pol II-containing complexes co-immunoprecipitated with mAb 8WG16. Proteins detected in the complexes affinity-purified with rabbit anti-SRm300 pAb are also listed in TABLE ONE if they (i) were detected by one or more spectra that overlapped with the proteins listed in TABLE ONE for the SRm160 purifications or (ii) were detected by three or more spectra irrespective of their detection in the SRm160 purifications. Although these criteria for specificity probably enrich for factors that form bona fide interactions with SRm160/300, they potentially also exclude specific but less strong interactions or factors that form specific and dual interactions with SRm160/300- and pol II-containing complexes.

Pre-mRNA Processing Factors Associated with SRm160-containing Complexes—Consistent with the known functional activities of SRm160 (see the Introduction), the majority of the proteins that interacted with SRm160/300-containing complexes by the above criteria are functionally linked to pre-mRNA processing. These included many spliceosomal proteins such as SF3b and U5 small nuclear ribonucleoprotein (snRNP)-associated proteins as well as proteins that remained associated with spliced mRNA in the exon junction complex (SRm160, Y14, and eukaryotic initiation factor 4AIII) (TABLE ONE and supplemental Table 1). Moreover, in agreement with recent evidence from our laboratory indicating that SRm160 participates in 3'-end processing (7, 8), we also detected four 3'-end cleavage factors in the immunopurified complexes, although with fewer peptide spectra per protein (supplemental Table 1).

Of particular interest among the spliceosomal proteins detected are the multiple interactions with subunits of the U2 snRNP-associated SF3b complex, including SAP145, SAP130, and SAP49 (TABLE ONE and supplemental Table 1). These factors are known to form a sequence-independent interaction upstream of the pre-mRNA branch region (the "anchor site") and to participate in formation of a pre-spliceosomal complex containing U1 and U2 snRNPs (32, 33). Taken together with our previous evidence that (i) SRm160 associates specifically with a subpopulation of U2 snRNPs in the absence of pre-mRNA (4), (ii) U2 snRNP promotes the stable association of SRm160 with pre-mRNA (4), and (iii) SRm160 promotes enhancer-dependent splicing via interactions that appear to be independent of the binding of U2AF to the polypyrimidine tract (19), the interactions detected with SF3b subunits suggest that SRm160 could associate with pre-mRNA via one or more interactions involving these proteins. This finding therefore provides new insight into the possible network of protein-protein interactions by which SRm160 functions as a splicing coactivator.

Transcription and Chromatin Regulatory Factors Associated with SRm160-containing Complexes—In addition to pre-mRNA processing components, many of the factors found to associate with SRm160/300-containing complexes are involved in pol II transcription and chromatin regulation. Moreover, some of these factors, including the far upstream element-binding protein FBP and its associated repressor protein RoBP1, have already been associated functionally with splicing as well as transcription (34, 35). The SR-related protein CAPER, which has recently been implicated in the regulation of alternative splicing via interactions with nuclear hormone receptor transcriptional activators (36), was also detected in SRm160/300-containing complexes, as were the nuclear hormone receptor (co)activator protein CIA (37) and the transcriptional regulator CCR4-NOT (38) (TABLE ONE). These results raise the possibility that SRm160/300 may function in the coupling of transcription and splicing via one or more interactions involving promoter-bound (co)activators and that this activity could be largely independent of direct interactions with pol II. In addition, several subunits of the NuRD chromatin remodeling complex (39–42), including SWI/SNF-170, SWI/SNF-155, Mi-2{beta}, MCDB3, and Mta2, co-immunoprecipitated with SRm160/300, although they were detected with fewer than three peptide spectra per protein (supplemental Table 1).

SRm160 Associates with Subunits of Cohesin—The immunoaffinity-purified SRm160-containing complexes also include four subunits of the human cohesin complex: SMC1{alpha}/SMC1L1, SMC3, hRAD21, and SA2 (TABLE ONE and supplemental Table 1). These four subunits of cohesin, which function in both sister chromatid cohesion and DNA recombination and repair activities, are known to form a complex in the absence of chromatin. However, proteins that associate with SMC1 and SMC3 in a DNA recombination/repair complex (RC1), including DNA ligase III, DNA polymerase {epsilon}, and DNA endonuclease (43–45), the SA1 subunit of cohesin (46, 47), or subunits of the DNA damage-induced BRCA1/SMC1/SMC3-containing complex (48, 49), were not identified by mass spectrometry in the immunoaffinity-purified SRm160-containing complexes. This suggests that SRm160 could form a specific association with the cohesin complex containing SMC1{alpha}, SMC3, hRAD21, and SA2.

We confirmed the co-immunoprecipitation of SMC1{alpha}, SMC3, and hRAD21 with SRm160 by immunoblot analysis with specific antisera (Fig. 1B; see "Experimental Procedures"). Approximately 10–15% of SMC1{alpha}, SMC3, and hRAD21 in the extract immunoprecipitated with mAb B1C8 (Fig. 1B, lane 3). The interaction of the cohesin subunits with SRm160 appears to be quite specific based on the various controls described above as well the observation that abundant proteins in the nuclear extract (e.g. the U1–70K protein) (Fig. 1B) were not detected in the mAb B1C8 immunoprecipitates by immunoblotting and were not detected by immunoblotting of the immunoprecipitates collected with rabbit anti-SRm300 pAb or mAb 8WG16 (Fig. 1B), whereas these latter antibodies did immunoprecipitate the expected proteins (e.g. mAb 8WG16 immunoprecipitated transcription factor TFIIF p74) (Fig. 1B, lanes 4 and 5, and TABLE ONE and supplemental Table 1). The absence of detectable co-immunoprecipitation of the cohesin subunits with rabbit anti-SRm300 pAb suggests that these proteins were bound to the subpopulation of SRm160 in the nuclear extract that was not associated with SRm300. Immunoprecipitates collected with rabbit anti-SRm160 pAb, which binds to a separate region in SRm160, were immunoblotted with the anti-SMC1{alpha} antibody. Similar to the results with mAb B1C8, a comparable level of SMC1{alpha} in the extract was co-immunoprecipitated with rabbit anti-SRm160 pAb (Fig. 1C, compare lane 4 with lanes 5–8), thus providing strong evidence that the co-immunoprecipitation of cohesin subunits with anti-SRm160 antibodies is due to an association between cohesin and SRm160 and not a cross-reaction between mAb B1C8 and one or more of the cohesin subunits. Finally, the association between SRm160 and cohesin subunits is unlikely a consequence of bridging interactions mediated by nucleic acid because the HeLa nuclear extract used for immunopurifications was pretreated extensively with both RNase and DNase using a protocol that efficiently degrades the most stable nucleic acids (e.g. U5 small nuclear RNA) (7) (data not shown).

Because cohesin is essential for sister chromatid cohesion and chromosome segregation, we considered that the association with SRm160 might be dependent on the stage of the cell cycle. We prepared extracts from HeLa cells synchronized in S or M phase by double thymidine block and release and analyzed the interaction of SRm160 with SMC1 and hRAD21 at different stages of the cell cycle (Fig. 1D). The stage of the cell cycle was confirmed by immunoblot analysis of the extracts with an antibody directed against phosphorylated Tyr15 in Cdc2, which is dephosphorylated upon entry into M phase (Fig. 1D, lower panel, lanes 2 and 3). Consistent with an earlier report (44), the level of SMC1 did not change throughout the cell cycle (Fig. 1D, lower panel, lanes 4 and 5). The relative levels of co-immunoprecipitation of SMC1{alpha} and hRAD21 with SRm160 were similar in G1/S phase, M phase, and asynchronous extracts, with ~15% of the input material being recovered in each case (Fig. 1D, upper panel, compare lanes 1–3 with lanes 4–9).



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  		FIGURE 2.
A subfraction of SRm160 co-fractionates with cohesin subunits. A, immunoprecipitations were performed using control beads (lanes 3, 5, 7, 9, and 11) or mAb B1C8 (lanes 4, 6, 8, 10, and 12) in buffer containing NaCl at the concentrations indicated. Loads 1 and 2 represent 3 and 0.7% of the input to the immunoprecipitation (lanes 1 and 2, respectively). B, HeLa nuclear extract was fractionated over a Sephacryl S400 column as described under "Experimental Procedures," and the elution fractions were analyzed by immunoblotting with the antibodies indicated. gAb-SRm160, goat anti-SRm160 pAb; TFIIF, transcription factor IIF; Sm, common snRNP Sm proteins.

 
Characterization of SRm160-Cohesin Interactions—Cohesins have been highly purified by immunopurification and chromatographic separation, and their associations with other factors have been characterized (46, 50–55); SRm160 was not identified as an interacting component in these previous studies. One possible reason for this is that the association of SRm160 with cohesin subunits is salt-sensitive. This is supported by the observation that cohesin subunits co-immunoprecipitated with SRm160 at physiological salt concentrations (Fig. 2A, lanes 3–8), but that these associations were markedly reduced at salt concentrations above 200 mM (lanes 9–12). Based on this observation, chromatographic purifications of cohesin involving high salt elution or ammonium sulfate precipitation, as used in previous studies (51, 53), would not be expected to contain SRm160.

To investigate whether the cohesin subunits exist in association with one or more distinct SRm160-containing complexes, we fractionated HeLa nuclear extract at 100 mM salt by gel filtration after extensive treatment with RNase and DNase and analyzed the fractions by immunoblotting with the different antisera to SRm160 as well as with antisera to several of the SRm160-associated factors (Fig. 2B). Interestingly, SRm160 was detected primarily in two different elution peaks, one centering on fraction 22 and the other centering on fraction 28. A form of SRm160 with slightly slower electrophoretic mobility, peaking in fraction 28, was detected more readily by mAb B1C8 and the murine anti-SR protein mAb 104, which was previously shown to also detect SRm160 (4). This can be explained by the observation that both mAb B1C8 and mAb 104 recognize epitopes that are phosphatase-sensitive (54).3 Both rabbit anti-SRm160 pAb (described above) and a peptide-specific goat anti-SRm160 antibody (see "Experimental Procedures") detected a protein of identical mobility in this region of the gradient as well as the more prominent, faster migrating form concentrated in fraction 22 (Fig. 2B) (data not shown). The faster migrating form most likely corresponds to SRm160 with a reduced phosphorylation status because previous experiments have also demonstrated that phosphatase treatment of nuclear extract or purified endogenous SRm160 significantly reduces the mobility of the protein as detected with rabbit anti-SRm160 pAb (28).3

Interestingly, the SMC1{alpha}, SMC3, and hRAD21 cohesin subunits were predominantly concentrated in fractions 28–30, whereas SR protein splicing factors were predominantly concentrated in fraction 22 (Fig. 2B). Although the mAb B1C8-detected form of SRm160 did not co-fractionate precisely with the cohesin subunits, this is consistent with its having a less stable association with these factors compared with their association with each other (Fig. 2A) and with the fact that only 10–15% of the total cohesin in nuclear extract associated with SRm160 during co-immunoprecipitation. This conclusion was further supported by the observation that SRm160 and cohesin displayed more extensively separated elution profiles when gel filtration chromatography of HeLa nuclear extract was repeated at a higher salt concentration (500 mM). Together, the results therefore suggest that a more highly phosphorylated form of SRm160 (detected preferentially by mAb B1C8 and mAb 104) preferentially co-fractionates with cohesin subunits, whereas a less phosphorylated form (more strongly detected with the anti-SRm160 pAbs) predominantly coelutes with SR protein splicing factors and not with cohesin subunits (data not shown). Thus, it is possible that differential phosphorylation modulates interactions between SRm160 and different complexes and, consequently, its potential to function in different cellular processes.

Interactions between SRm160 and Cohesin Subunits Are Evolutionarily Conserved—If the association between SRm160 and cohesin subunits is functionally important, we would expect to detect it in other species as well. To test this, we assayed whether mAb B1C8 co-immunoprecipitates SMC1{alpha} in X. laevis egg extract. SRm160 is highly conserved in metazoans, especially within the N-terminal region of the protein that includes the PWI motif (8). The Xenopus tropicalis open reading frame (GenBankTM accession number BC074646 [GenBank] ) shares 85% similarity and 78% identity (data not shown). mAb B1C8 was shown previously to immunoprecipitate exon junction complexes containing SRm160 that form on spliced mRNA in Xenopus egg extracts (6, 55). Detection of a 220-kDa antigen in these studies is consistent with SRm160 having a longer predicted open reading frame in Xenopus than in human (954 versus 820 amino acids). Moreover, we detected a prominent 220-kDa antigen in X. laevis egg extract with both mAb B1C8 and rabbit anti-SRm160 pAb (Fig. 3A, lanes 2, 3, 5, and 6) (data not shown). Rabbit anti-SRm160 pAb also recognized bands of ~160 and ~140 kDa in Xenopus extract, which presumably correspond to different phosphoisoforms of SRm160. The detection of the 220-kDa antigen by rabbit anti-SRm160 pAb in immunoprecipitates collected from Xenopus egg extract with mAb B1C8 confirmed that these antibodies bound to the same 220-kDa antigen (Fig. 3A, lanes 5 and 6). Together, these results provide strong evidence that our antisera to hSRm160 specifically detect the Xenopus ortholog of SRm160. Like SRm160, SMC1{alpha} is highly conserved in Xenopus (GenBankTM accession number O93308 [GenBank] ), sharing 85% identity over 1233 amino acids, and the anti-hSMC1 antibody detected an antigen in Xenopus egg extract of identical mobility to the human protein (Fig. 3B, compare lanes 1 and 3), which has been confirmed as Xenopus SMC1 (55). Similar to the results in HeLa nuclear extract, mAb B1C8 co-immunoprecipitated 10–20% of total SMC1 in Xenopus egg extract (Fig. 3C, compare lanes 3 and 4 with lanes 5 and 6). This indicates that the interaction between SRm160 and SMC1 is conserved between distantly related vertebrate species.



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  		FIGURE 3.
Interaction between SRm160 and cohesin is conserved in Xenopus. A, immunoblot analysis of 30 µg of HeLa nuclear extract (NE; lanes 1 and 4) and 1 µl (100 µg; lanes 2 and 5) and 2 µl (200 µg; lanes 3 and 6) of Xenopus egg extract (Ex) probed with mAb B1C8 (lanes 1–3) and rabbit anti-SRm160 pAb (rAb {alpha}SRm160; lanes 4–6). x, Xenopus. B, immunoblot analysis of HeLa nuclear extract and Xenopus egg extract as described for A using the anti-SMC1 antibody. C, co-immunoprecipitation of SMC1 with SRm160 from HeLa nuclear extract (lanes 1, 3, and 4) and Xenopus egg extract (lanes 2, 5, and 6) using mAb B1C8. Immunoprecipitates were analyzed by immunoblotting and probed with the anti-SMC1 antibody and rabbit anti-SRm160 pAb. The load represents 5% of the input amount used in the immunoprecipitations. The asterisk denotes a probable SRm160-containing aggregate, which we detected occasionally in HeLa extracts.

 
A Genetic Interaction between SRm160 and Cohesin in C. elegans—RNAi was used next to investigate a possible functional relationship between SRm160 and the cohesin complex in the context of animal development. C. elegans provides a convenient model organism to address this question because the CeSRm160 ortholog (rsr-1) is highly conserved, sharing 39% identity and 48% similarity with mammalian SRm160 (56). Furthermore, because CeSRm160 is a nonessential protein (24), it is possible to investigate which other nonessential factors interact with it genetically using combinatorial RNAi. Cohesin subunits are represented by several orthologs in C. elegans that share considerable sequence identity, and previous experiments using RNAi have established that these proteins function in the same pathways as they do in mammals (supplemental Table 2).

Previous combinatorial RNAi experiments have also established that simultaneous RNAi of CeSRm160 and different CeSR proteins or 3'-end cleavage factors results in specific developmental defects, whereas single RNAi of these factors in most cases does not result in apparent phenotypes (11, 24, 56). The specificity of the combinatorial RNAi phenotypes observed in these previous studies most likely reflects the specificity of conserved interactions and functional associations observed between human SRm160, SR, and cleavage proteins. Thus, combinatorial RNAi of CeSRm160 and conserved partner proteins, identified by biochemical assays in the mammalian system, can provide useful information on the potential role of conserved interactions involving SRm160 in the context of whole animal development.

RNAi of the orthologs of most C. elegans cohesin subunits results in early embryonic lethal phenotypes (57–59); these genes therefore cannot be tested for genetic interactions with SRm160. However, RNAi of coh-1 (the hRAD21 homolog) has been reported to produce a weak uncoordinated phenotype (supplemental Table 2). We therefore determined whether simultaneous RNAi of CeSRm160 and coh-1 results in an exacerbated phenotype. Interestingly, an embryonic lethal phenotype (18% of animals) and a strong uncoordinated phenotype were observed (TABLE TWO). Simultaneous RNAi of coh-1 and either of the splicing factors CeSRp75 and CeRNPS1, which are known to associate with a subpopulation of SRm160 in human extracts (11, 56), did not result in an altered phenotype because the majority of the animals were weak uncoordinated (TABLE TWO). These results suggest that SRm160 is functionally linked to the cohesin complex and that an interaction between these factors may be important for animal development.


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  		TABLE TWO
Phenotypes associated with combinatorial RNAi of coh-1 and splicing-related genes

 
SRm160 Is Present in Meiotic Synaptonemal Complexes—The conserved interaction between SMC1{alpha}, SMC3, and hRAD21 with a subpopulation of SRm160 in nuclear extract and the observation of a combinatorial RNAi phenotype between SRm160 and coh-1 in C. elegans strongly suggest that SRm160 exists in one or more functional complexes with cohesins in vivo. To further investigate this, we determined whether SRm160 localizes at cohesin-containing meiotic synaptonemal complexes (SCs) that form between paired homologous chromosomes because these can be readily visualized by microscopy in mouse spermatocytes at the pachytene stage of meiotic prophase I. Using this system, it has been shown that meiosis-specific cohesin subunits (which include the SRm160-interacting subunit SMC3) are localized to fiber-like structures along the SCs, which correspond to cohesin cores co-localizing with axial elements (60). Moreover, it has been shown that SMC1{beta} is required for normal progression of prophase I in mouse spermatocytes (60). The cohesin cores co-localize precisely with SCP3/COR1 (61), a core SC component located primarily in the axial elements; and antibodies to this factor, which perform well in immunolocalization studies, were therefore used as a marker for the cohesin cores.

Immunoblot analysis of total (asynchronous) spermatocytes and spermatocytes synchronized at the diplotene and pachytene stages with both rabbit anti-SRm160 pAb and mAb B1C8 resulted in the detection of an ~180-kDa antigen (Fig. 4A, lanes 2–5) (data not shown). Although a minor 180-kDa form of SRm160 has been detected by mAb B1C8 in human cell extracts (e.g. Fig. 2B) (9), the more prominent detection of an antigen of this size in mouse extracts could reflect differences in the phosphorylation status of SRm160 and/or the increased size of the mouse protein compared with the human protein (897 versus 820 amino acids) as predicted from a full-length mouse cDNA sequence (GenBankTM accession number NM_016799 [GenBank] ). Immunostaining of the mouse spermatocytes with mAb B1C8 (Fig. 4B, upper panels) and antigen affinity-purified rabbit anti-SRm160 pAb (lower panels) revealed that both antibodies strongly labeled SCs that formed between paired autosomes, as revealed by co-immunostaining with murine monoclonal and rabbit polyclonal antisera specific for SCP3/COR1 (Fig. 4B, upper and lower panels, respectively). (Note that the immunostainings with rabbit anti-SRm160 and anti-SCP3/COR1 pAbs were slightly displaced in the overlays to more readily afford direct comparison of the individual staining patterns.) An antiserum specific for the centromeres was also included in the immunostainings, and these sites are indicated. The control rabbit anti-glutathione S-transferase pAb did not label these or any other structures in the spreads (data not shown). The localization of SRm160 to SCs associated with unpaired X and Y cores (Fig. 4B) suggests that SRm160 is closely associated with the cohesin core structures that form part of the SC axial elements. The localization of SRm160 to SCs is consistent with its having a possible functional association with cohesin during homologous chromosome pairing and recombination during meiosis.



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  		FIGURE 4.
SRm160 is localized in synaptonemal complexes. A, samples (2–5 x 106 cell eq) derived from HeLa nuclear extract (lane 1), mouse spermatocytes (lane 2), diplotene stage spermatocytes (lane 3), pachytene spermatocytes (lane 4), and diplotene spermatocytes (lane 5) were analyzed by SDS-PAGE (7.5% gel), followed by immunoblotting with rabbit anti-SRm160 pAb (rAb {alpha}SRm160). mSRm160, mouse SRm160. B, shown is the immunofluorescence of mouse spermatocyte nuclei probed with mAb B1C8 (upper panels) or rabbit anti-SRm160 pAb (lower panels) and rabbit or mouse anti-SCP3/COR1 antibody. SCs, pseudo-autosomal regions (PAR) of the X (X) and Y (Y) chromosomes, and centromeres (CEN) are indicated. Note that the immunostainings with rabbit anti-SRm160 and anti-SCP3/COR1 pAbs were slightly displaced in the overlays to more readily afford direct comparison of the individual staining patterns.

 
Conclusion—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. Several of these interactions suggest previously unanticipated functions for SRm160. 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.

Our observation that core subunits of a cohesin complex form conserved associations with a subpopulation of SRm160 indicates that these factors participate in one or more common cellular functions. Interestingly, previous mass spectrometric analyses of purified splicing complexes revealed SMC1 and SMC2 in pre-spliceosomes (62, 63), although the functional significance of these interactions was not investigated. Moreover, extensive mutagenesis screens for splicing factors in budding yeast have not yielded any pre-mRNA processing gene mutants that correspond to cohesin subunit genes, which nevertheless are highly conserved in this organism (64). Our identification of interactions between SRm160 and SMC1{alpha}, SMC3, hRAD21, and SA2 and the localization of SRm160 to core regions of SCs argue that it is more likely that SRm160 functions with cohesin in the context of one or more activities associated with chromatin structure and/or sister chromatid cohesion. The presence of a highly conserved PWI motif in SRm160, which can bind both single- and double-stranded DNA and RNA (8), further suggests a possible separate role for SRm160 in DNA metabolism, in addition to its functions in pre-mRNA processing. Finally, the developmental defect observed in C. elegans upon simultaneous RNAi of coh-1 and CeSRm160, but not upon simultaneous RNAi of coh-1 and other C. elegans splicing factor orthologs (CeSRp75 and CeRNPS1), also supports a separate functional role for SRm160 in association with cohesins.


   FOOTNOTES
 
* 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. Back

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

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.

2 The abbreviations used are: h, human; RNAi, RNA interference; Ce, C. elegans; mAb, monoclonal antibody; pAb, polyclonal antibody; pol II, RNA polymerase II; snRNP, small nuclear ribonucleoprotein; SCs, synaptonemal complexes. Back

3 S. McCracken and B. J. Blencowe, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Jan Michael Peters, Bill Earnshaw, Claudio Sunkel, Angus Lamond, Joan Steitz, Mark Roth, and Jack Greenblatt for the generous gifts of antibodies. We thank Iain Johnstone (University of Glasgow) for the use of the microinjection facility. We also thank Ana Losada and Peter Roy for help with some of the experiments and May Khanna, Arneet Saltzman, John Calarco, Joanna Ip, and Emanuel Rosonina for helpful suggestions and proofreading the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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