RBM5/Luca-15/H37 Regulates Fas Alternative Splice Site Pairing after Exon Definition

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1 Article RBM5/Luca-15/H37 Regulates Fas Alternative Splice Site Pairing after Exon Definition Sophie Bonnal, 1 Concepción Martínez, 1,5 Patrik Förch, 4,6 Angela Bachi, 4,7 Matthias Wilm, 4,8 and Juan Valcárcel 1,2,3, * 1 Centre de Regulació Genòmica 2 Institució Catalana de Recerca i Estudis Avançats 3 Universitat Pompeu Fabra Dr. Aigüader, Barcelona, Spain 4 European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany 5 Present address: European Molecular Biology Laboratory Monterotondo, Adriano Buzzati-Traverso Campus, Monterotondo, Italy 6 Present address: UCB S.A., Chemin du Forest, B-1420 Braine L Alleud, Belgium 7 Present address: San Raffaele Research Institute, Via Olgettina 60, Milano, Italy 8 Present address: Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, 4 Dublin, Ireland *Correspondence: juan.valcarcel@crg.es DOI /j.molcel SUMMARY RBM5/Luca-15/H37 is a gene frequently inactivated in lung cancers and overexpressed in breast tumors. Its protein product has been detected in prespliceosomal complexes and modulates cell proliferation and Fas-mediated apoptosis. We report that RBM5 is a component of complexes involved in 3 0 splice site recognition and regulates alternative splicing of apoptosis-related genes, including the Fas receptor, switching between isoforms with antagonistic functions in programmed cell death. In contrast with classical mechanisms of splicing regulation, RBM5 does not affect early events of splice site recognition that lead to Fas exon 6 definition. Instead, RBM5 inhibits the transition between prespliceosomal complexes assembled around exon 6 to mature spliceosomes assembled on the flanking introns and promotes sequence-specific pairing of the distal splice sites. An OCRE domain important for RBM5 function contacts components of the U4/5/6 tri-snrnp, consistent with the idea that RBM5 modulates splice site pairing after prespliceosome assembly and exon definition. INTRODUCTION Deletion of chromosomal region 3p21.3 is the most frequent early genetic alteration in lung cancer. This deletion is also common in lung tissue from smokers and in carcinomas from other tissues (Lerman and Minna, 2000; Wistuba et al., 1997). Allelic loss of this region is often followed by inhibition of expression of the remaining alleles, a phenomenon affecting the putative tumor suppressor gene RBM5, also known as LUCA-15 or H37, which is silenced in 70% 80% of lung cancers (Wei et al., 1996; Edamatsu et al., 2000; Timmer et al., 1999; reviewed by Sutherland et al., 2005; Maarabouni and Williams, 2006). Downregulation of RBM5 also occurs in other cancers, upon oncogenic Ras activation, in cell lines and is one molecular signature associated with metastasis in various solid tumors (Edamatsu et al., 2000; Welling et al., 2002; Ramaswamy et al., 2003). By contrast, overexpression of the oncogene HER-2/neu, which is a frequent feature of breast and ovarian tumor progression, leads to RBM5 upregulation in cells derived from breast and ovarian cancers (Oh et al., 1999). Increased levels of RBM5 have, indeed, been detected in breast cancer samples (Oh et al., 1999; Rintala- Maki et al., 2007). These observations suggest that both decreased and increased levels of RBM5 can play a role in tumor progression. Overexpression of RBM5 major isoform inhibits cell proliferation by extending the G1 phase of the cell cycle in tumor cell lines (Edamatsu et al., 2000; Mourtada-Maarabouni et al., 2003). Expression of RBM5 in various cell lines reduces their potential to form colonies and to induce tumors when transferred into nude mice (Oh et al., 2002). Increased levels of RBM5 promote apoptosis in cell lines from various origins, mediated through several pathways, including CD95(Fas), TNF-a, and TRAIL (Rintala-Maki and Sutherland, 2004; Rintala-Maki et al., 2004). Therefore, RBM5 can influence both cell division and apoptosis. RBM5 transcripts encode a protein of 815 amino acids with sequence motifs characteristic of factors involved in premrna splicing and other aspects of RNA metabolism (Sutherland et al., 2005). These include two RNA recognition motifs (RRM), two zinc fingers, one arginine-serine (RS) domain, and one glycine (G) patch (Figure 1E). The protein also contains an OCtamer REpeat (OCRE) domain of unknown function, consisting of repeats of eight residues organized around a triplet of aromatic amino acids, which is present in some RBM proteins and in the angiogenic factor VG5Q (Callebaut and Mornon, 2005). This isoform has been recently identified as a component of prespliceosomal complexes (Deckert et al., 2006; Behzadnia et al., 2006). Three alternatively spliced RBM5 mrnas can encode truncated proteins (Sutherland et al., 2005) that antagonize the functions of the full-length protein, facilitating cell proliferation and inhibiting apoptosis (Mourtada-Maarabouni et al., 2003). An antisense transcript of the locus has also been shown to antagonize the proapoptotic functions of the full-length RBM5 isoform (Sutherland et al., 2000; Mourtada-Maarabouni et al., 2002). Molecular Cell 32, 81 95, October 10, 2008 ª2008 Elsevier Inc. 81

2 Figure 1. U2AF65/RBM5 Interactions (A) Identification of U2AF 65 -associated proteins. Immunoprecipitates using anti-u2af 65 or control antibodies were washed with buffer containing the indicated concentrations of NaCl. Proteins eluted from the pellet were analyzed by electrophoresis on denaturing SDS-polyacrylamide gels and Colloidal Coomassie Blue staining. M1, M2: molecular weight markers. The identity of the proteins indicated, determined by mass spectrometry, can be found in Table S1. (B) RBM5 immunoprecipitates contain U2AF 65. Precipitates of HeLa nuclear extracts with anti-rbm5 or control antibodies were analyzed by western blot using the MC3 anti-u2af 65 antibody with or without treatment with RNase. (C) Direct interaction of RBM5 and U2AF 65. GST-U2AF 65 (containing or lacking its N-terminal RS domain, residues 1 92) and His-RBM5 were incubated in buffer D 0.1 M KCl, and, after pull-down using glutathione beads, His-RBM5 was detected in the pellet by electrophoresis in SDS gels and western blot analysis. (D) Coimmunoprecipitation of U2AF 65 and T7-epitope-tagged RBM5 (full-length or deletion mutants) expressed in 293T cells. Total cell extracts of transfected cells were precipitated with anti-u2af65 (MC3) or control antibodies and the precipitates analyzed by western blot using anti-t7 antibodies. DC-terminal and DOCRE correspond to deletion of residues and of RBM5, respectively. (E) Arrangement of protein motifs in the primary structure of RBM5. The molecular mechanisms of RBM5 function are still poorly understood. Extended G1 correlated with reduced levels of cyclin A and of phosphorylated Rb (Oh et al., 2006). Induction of apoptosis has been correlated with altered levels of Bcl-2, Bclx(L), and Bax (Sutherland et al., 2001; Mourtada-Maarabouni et al., 2002; Oh et al., 2006). Up- or downregulation of RBM5 cause changes in the transcript levels of about 35 genes related with the control of cell proliferation and apoptosis (Mourtada- Maarabouni et al., 2006). It is, however, unclear whether any of these factors are direct targets of RBM5 and what the underlying regulatory mechanisms may be. Regulation of apoptosis has not been rigorously linked to specific splicing regulators. In this manuscript, we identify RBM5 as a regulator of alternative splicing that switches between functionally distinct isoforms of the Fas receptor and the Fas pathway regulator c-flip. 82 Molecular Cell 32, 81 95, October 10, 2008 ª2008 Elsevier Inc.

3 Alternative pre-mrna splicing significantly contributes to the proteomic diversity of multicellular organisms (Ast, 2005; Blencowe, 2006; Ben-Dov et al., 2008), affecting at least 74% of human multiexon genes (Blencowe, 2006), generating up to thousands of protein isoforms from the same gene, which can harbor distinct and sometimes antagonistic functions. A significant fraction of genetic diseases is caused by alterations in the splicing process, and misregulation of alternative splicing is known to cause or modify human pathologies (Wang and Cooper, 2007). Despite the incidence and biological relevance of alternative splicing, the molecular mechanisms controlling this process and their alterations in pathological situations are not well understood. Removal of intronic sequences is carried out by the complex molecular machinery of the spliceosome, which includes five small nuclear ribonucleoprotein particles (snrnps) and more than 200 polypeptides (Will and Lührmann, 2005). Early events in the recognition of splicing signals include the binding of U1 snrnp to the 5 0 splice site and of the branch point binding protein BBP/SF1 and the two subunits of the splicing factor U2AF to the branch point, polypyrimidine tract, and conserved AG located at the 3 0 end of the intron (Will and Lührmann, 2005). Direct or indirect bridging interactions between these components across the intron are thought to commit splice sites for intron removal. Initial recognition of the splice sites flanking internal exons, which are typically much shorter than the surrounding introns, occurs through similar stabilizing interactions by the process of exon definition (Berget, 1995). Regulation of splice site utilization often targets these early events in splice site recognition. For example, proteins of the SR and hnrnp families of splicing regulators have been shown to facilitate or prevent access of U1 snrnp or U2AF to their cognate sequences (Blencowe, 2006). The protein PTB has been shown to induce skipping of regulated exons by inhibiting exon and intron definition (Izquierdo et al., 2005; Sharma et al., 2005). Replacement of BBP by U2 snrnp at the branch point site leads to the ATP-dependent assembly of prespliceosomal complexes (also known as complex A). Incorporation of the U4/5/6 tri-snrnp and concomitant rearrangements of snrna-premrna, snrna-snrna, and protein-rna interactions leads to fully assembled spliceosomes where the two-step reaction of intron removal and exon ligation takes place (Will and Lührmann, 2005). A poorly understood step in the splicing process is how the initial stabilizing interactions across the exon that lead to exon definition are switched to the interactions between the splice sites across the introns that are involved in intron removal. In this manuscript, we report that RBM5 regulates Fas exon 6 alternative splicing by targeting this switch, thus acting as a selector of splice site pairing after exon definition. RESULTS Identification of U2AF 65 -Associated Proteins U2AF 65 was immunoprecipitated from HeLa cell nuclear extracts using monoclonal antibody MC3 (Gama-Carvalho et al., 1997). Coprecipitated polypeptides were eluted from the pellet by washing the precipitate with increasing salt concentrations (Figure 1A). About 20 polypeptides were specifically eluted from U2AF 65 precipitates at 500 mm NaCl (Figure 1A, compare lanes 7 and 8) and identified by MALDI-TOF mass-spectrometry (Table S1 available online). In addition to U2AF 35, which was eluted from the precipitates together with U2AF 65 using SDS, the components of the complex include: (1) two isoforms of SF1/BBP; (2) components of U1 snrnp (U1 70K) and U2 snrnp (including U2A, U2A 0, U2B 00, and components of the Sm, SF3a, and SF3b complexes), suggesting that at least a fraction of U2AF is associated with U2 snrnp in nuclear extracts; (3) the 3 0 splice site recognizing factor SPF45; (4) spliceosomal components of unknown function, including SPF31 and the longer isoform of the putative tumor suppressor gene RBM5/LUCA-15/ H37; (5) proteins of the hnrnp family of RNA-binding factors; and (6) other proteins with sequence features characteristic of factors involved in RNA metabolism. This manuscript focuses on the product of the putative tumor suppressor gene RBM5/LUCA-15/H37. Antibodies generated against the purified recombinant full-length RBM5 protein detected a single polypeptide of 110 kda in nuclear extracts, consistent with previous reports (Rintala-Maki and Sutherland, 2004) (Figure S1). Immunoprecipitates of RBM5 using these antibodies contained U2AF 65 (Figure 1B), confirming the association between the two proteins. This association did not require RNA (Figure 1B), was also detectable in pull-down assays using recombinant purified proteins, and required the amino-terminal RS domain of U2AF 65 (Figure 1C). The association with U2AF 65 complexes was also detected in extracts from 293T cells expressing epitope-tagged full-length RBM5, but not a mutant lacking the carboxy-terminal region of RBM5, suggesting that neither the RS nor the RRM domains of the protein are sufficient to mediate the incorporation of RBM5 into U2AF complexes (Figure 1D). Immunolocalization and live fluorescent microscopy results were also consistent with RBM5 colocalization with splicing factors and U2AF 65 (Figure S2). RBM5 Regulates Alternative Splicing of Apoptotic Genes Because several results have implicated RBM5 in the regulation of Fas-mediated apoptosis, experiments were carried out to test the possibility that RBM5 regulates alternative splicing of genes involved in the control of apoptosis. RBM5 was knocked down by RNAi, and alternative splicing of several apoptosis-related genes, including Fas, c-flip, caspase 9, Mcl-1, and Blc-x, was analyzed by quantitative RT-PCR using isoform-specific primers. Initial experiments failed to observe changes in alternative splicing in cells in which RBM5 was depleted. The similarity between RBM5 and the products of the genes RBM6 and RBM10 (30% and 50% identity) suggested the possibility that these proteins display overlapping activities and, therefore, that depletion of more than one family member was necessary to observe alternative splicing changes. Subsequent experiments supported this concept. Depletion of RBM5, 6, and 10 resulted in alternative splicing changes in Fas receptor exon 6 and c-flip regulatory factor exon 7 (Figure 2A). Fas exon 6 can be included or skipped under different cellular conditions, leading to the synthesis of either a mrna encoding the membrane-bound, proapoptotic form of the Fas receptor or a soluble form that is known to act as an inhibitor of apoptosis (Cheng et al., 1994; Molecular Cell 32, 81 95, October 10, 2008 ª2008 Elsevier Inc. 83

4 Figure 2. Regulation of Fas and c-flip Alternative Splicing by RBM5 (A) Silencing of RBM5, 6, and 10 results in changes in alternative splicing of Fas and c-flip genes. RNA was isolated from HeLa cells 72 hr after transfection with sirnas specific for RBM5, 6 and three isoforms of 10 or control sirnas (Table S4). After reverse transcription using oligodt and random primers, real-time PCR reactions were set up using the color-code indicated pairs of Fas/c-FLIP gene/isoform-specific oligonucleotides. Primers drawn over splicing patterns correspond to splice junction oligos, which can only hybridize with the corresponding spliced mrna. Changes in ratio between isoforms (represented as Log 2) were determined by measuring the relative abundance of alternatively spliced transcripts normalized by gene expression (see Supplemental Experimental Procedures). The values correspond to average and standard deviation of three independent experiments. (B) Increased skipping of endogenous Fas exon 6 upon RBM5 overexpression. RNA was purified from HeLa cells transfected with a plasmid expressing RBM5 and analyzed by RT-PCR using Fas-specific primers. The fraction of exon-skipped transcripts for three independent experiments is indicated. The change in exon skipping induced by RBM5 overexpression is indicated. Levels of RBM5 protein were monitored by western blot using specific antibodies. (C) RBM5 induces Fas exon 6 skipping from a minigene reporter. Fas genomic sequences between the 5 0 end of exon 5 and 47 first nucleotides downstream of the 5 0 splice site of exon 7 were cloned in an expression vector and transfected into HeLa cells together with a T7-RBM5 expression plasmid, a mutant derivative 84 Molecular Cell 32, 81 95, October 10, 2008 ª2008 Elsevier Inc.

5 Cascino et al., 1995). The soluble, antiapoptotic Fas isoform was reduced upon RBM5, 6, and 10 depletion. Alternative isoforms of the c-flip gene include c-flip(s), a strong inhibitor of the Fas pathway that heterodimerizes with procaspase 8 and prevents its proteolytic activation within the DISC complex (Krueger et al., 2001a), and c-flip(l), which exerts a more limited inhibition of procaspase 8 cleavage (Krueger et al., 2001b) and that has been reported to activate the Fas pathway (Chang et al., 2002; Micheau et al., 2002). Depletion of RBM5, 6, and 10 resulted in increased c-flip(s)/(l) ratios. To investigate the mechanisms behind the effects of RBM5, we focused on the alternative splicing event involving Fas exon 6. Consistent with the effects of RBM5, 6, and 10 depletion, overexpression (3 fold) of RBM5 in HeLa cells promoted skipping of exon 6 from endogenous Fas pre-mrna (Figure 2B) or from a minigene expressing Fas genomic sequences between exons 5 and 7 (Figure 2C). The effects of reducing the endogenous levels of RBM5 were analyzed using a Fas minigene harboring a mutation at the polypyrimidine tract upstream of exon 6 (U-20C), which leads to significant levels of exon skipping and, thus, allows detection of increased levels of exon inclusion. Increased inclusion of Fas exon 6 was observed upon knockdown of the expression of RBM5, 6, and 10 (Figures 2D, lanes 1 3, and 2E), the bulk of the activity being associated with depletion of RBM5 and 10 (Figures 2D, lanes 4 6, and S3B). The combined effect of depletion of these factors could be reversed by expression of a sirna-resistant form of RBM5 lacking the glutamine-rich domain (Figure 2D), which was found to be dispensable for RBM5 activity in overexpression experiments (Figures 2C and S3A), but significantly less so by exogenous expression of the wild-type protein, which can still be targeted by the sirna (Figure S4). The combined results of overexpression and gene knockdown experiments confirm that the levels of RBM5 influence the extent of Fas exon 6 inclusion. The data imply that RBM5 is a regulator of alternative pre-mrna splicing and suggest that this activity can be relevant for the function of the protein as a modulator of apoptosis. RBM5 Activity Requires Fas Exon 6 Sequences and an Associated Weak 3 0 Splice Site The effects of RBM5 resemble those of the splicing regulator PTB, which was found to promote exon 6 skipping by binding to a uridine-rich exonic silencer (URE6) (Figure 3A; Izquierdo et al., 2005). Three different mutations of the URE6 element did not compromise the response to RBM5 (Figure 3B, mutants m0, m1, and m2; Izquierdo et al., 2005). Exon 6 sequences, however, were required for RBM5 function because replacement of exon 6 by four other exons or control sequences (e123) significantly reduced the effects of RBM5 overexpression (Figure 3C). To delineate RBM5-responsive elements within exon 6, the exon sequence was divided in three segments of approximately the same length (e1, e2, and e3 in Figure 3A; e2 corresponds to the URE6 silencer). Each segment was replaced alone or in combination with the other elements. Replacement of sequences e1 (two different substitutions) and/or e2 (three different substitutions) did not compromise RBM5 function (Figures 3B and 3D, lanes 5 8). Replacement of the e3 element by three different sequences caused higher levels of exon 6 skipping (Figure 3D, lane 13, and data not shown), suggesting the presence of an exonic splicing enhancer in this region. The e3 region was further subdivided in three consecutive subsegments of similar length, e3.1, e3.2, and e3.3, which allowed mapping of the enhancer within 7 12 nucleotides in e3.1 e3.2 (Figure 3D, lanes 15, 17, and 19). Because mutation of this enhancer resulted in significant levels of exon skipping, it was difficult to assess whether the efficiency of RBM5-induced exon skipping was comparable to that observed with the wild-type construct. Combined mutation of the e3 enhancer and the e2 silencer (mutant e23, lanes 11 and 12) rendered levels of exon inclusion similar to the wildtype exon (lane 11), and the exon skipping-promoting effects of RBM5 were 50% reduced in this mutant (lane 12), suggesting that e3 sequences are important for full response to RBM5. Taken together, the results revealed that Fas exon 6 sequences are important for RBM5 function and that full repressive activity by RBM5 requires sequences with exonic splicing enhancer function located at the 3 0 third of exon 6. Next, the influence of splice site strength on regulation by RBM5 was analyzed. First, as observed for regulation by PTB, strengthening the 5 0 splice site associated with exon 6 by improving its base pairing potential with U1 snrna (mutant U1c) did not compromise RBM5 function (Figure 3E, lanes 1 6). Efficient recognition of this 5 0 splice site requires an adjacent uridine-rich intronic enhancer (URI6) (Izquierdo et al., 2005). Although mutation of the URI6 element (muri6) resulted in increased levels of exon skipping, weakening the 5 0 splice site did not abolish RBM5 function (Figure 3E, lanes 7 and 8). Second, the relevance of the strength of the competing 3 0 splice sites was analyzed. The 3 0 splice site associated with exon 6 and located in intron 5 contains a rather short and relatively uridinepoor polypyrimidine tract and is, therefore, predicted to be weak compared to the downstream 3 0 splice site located in intron 6. RBM5-induced exon 6 skipping was not affected by replacing the 3 0 splice site of intron 6 by that of intron 5, thus making both splice sites equally weak (Figure 3E, lanes 11 and 12). Regulation was significantly compromised, however, when the two 3 0 splice sites were swapped (lanes 13 and 14), suggesting that a strong 3 0 splice site upstream of exon 6 limits the response to RBM5. Full lacking the Q-rich domain (amino acids ), or T7-ADAR as control. RNA was isolated 24 hr after transfection and analyzed by RT-PCR using vector-specific sequences. Molecular weight markers and the position of the spliced products are indicated. Quantification of three independent experiments was carried out as in (B). Levels of RBM5/ADAR proteins were monitored by western blot using anti-t7 epitope antibodies. (D) Depletion of RBM5 and RBM10 increases Fas exon 6 inclusion. sirnas specific for RBM5, 6, and three isoforms of 10 (Table S4) were transfected into HeLa cells, which were transfected 48 hr later with a mutant Fas minigene containing a U-to-C substitution at intron 5 position 20 and a sirna-resistant form of RBM5 or empty vector. RNAs were analyzed as in (B). (E) Depletion of RBM5 and RBM10 levels by RNAi. Western blot analyses of protein extracts from samples in (D) analyzed using antibodies against RBM5 and RBM10 (v1 isoform) or a-tubulin as a control. Due to unavailability of RBM6-specific antibodies, depletion of RBM6 expression was verified by RT-PCR (data not shown). Molecular Cell 32, 81 95, October 10, 2008 ª2008 Elsevier Inc. 85

6 Figure 3. Exon 6 Sequences and a Weak Associated 3 0 Splice Site Are Required for RBM5-Mediated Regulation (A) Schematic representation of Fas genomic sequences and mutants used in this study. e1 e3 represent three segments of exon 6; e2 corresponds to the previously defined uridine-rich exonic silencer (URE6) that mediates PTB repression. URI6 corresponds to a uridine-rich intronic enhancer that mediates exon 6 inclusion by TIA-1, mutated in muri6. Various mutants replacing exon 6 sequences are represented by a single scheme. U1c corresponds to a mutant that increases base-pairing complementarity of exon splice site with U1 snrna. Mutant sequences are provided in Table S2. (B) URE6 silencer is not required for RBM5 function. Cotransfection assays and RNA analyses were carried out as in Figure 2C. m0, m1, and m2 represent different sequences replacing the URE6 silencer (Izquierdo et al., 2005). Values of changes in exon skipping induced by RBM5 are represented as average and standard deviation for a minimum of three independent experiments. (C) Exon 6 sequences are important for RBM5 function. Mutant minigenes replacing e1-to-e3 sequences (represented in [A] as a single construct with vertical bars) are indicated (sequences provided in Table S3) and analyzed as in (B). 86 Molecular Cell 32, 81 95, October 10, 2008 ª2008 Elsevier Inc.

7 regulation was also compromised when the sequence of intron splice site was present at both acceptors or when intron splice site was replaced by a strong polypyrimidine tract (Figure 3E, lanes 15 18). Taken together, these results argue that a weak 3 0 splice site flanking the regulated exon is required for efficient RBM5 function and that the optimal configuration of 3 0 splice sites involves a weak site in intron 5 and a stronger competing site in intron 6. RBM5 Inhibits Splicing of Introns 5 and 6 As a first step to understand the mechanism of RBM5 regulation, RNA was isolated from HeLa cells under conditions of RBM5 knockdown/overexpression, and splicing was analyzed by RT- PCR using primers designed to detect splicing or retention of each of the introns flanking exon 6. To allow detection of the longer intron 6, a minigene harboring an intron deletion that did not affect regulation by RBM5 (Figure S5) was used. These analyses revealed that increased levels of RBM5 resulted in detectable levels of retention of both intron 5 and intron 6 (Figure 4A). Conversely, RBM5 depletion resulted in enhanced splicing of each of the individual introns, an effect reversed by expression of sirnaresistant RBM5 (Figure 4B). The collective data indicate that RBM5 exerts a similar level of splicing repression on introns 5 and 6. To further understand the steps of the splicing process targeted by RBM5, in vitro splicing assays were carried out and analyzed by primer extension using splice junction primers as previously described (Izquierdo et al., 2005). Consistent with the effects observed in vivo, addition of recombinant RBM5 expressed in and purified from either E. coli, baculovirus-infected insect cells, or 293T cells resulted in inhibition of splicing of introns 5 and 6 (Figure 4C and data not shown), while the converse effect was observed either upon immunodepletion of RBM5 from extracts or upon immunoinhibition by addition of anti-rbm5 antibodies to the extract (Figure 4D). These results confirm that RBM5 plays a direct role in the splicing process. Intriguingly, while addition of PTB led to inhibition of introns 5 and 6 and a concomitant increase in the product of splicing generated by exon 6 skipping (Figure 4C, lane 3), RBM5 did not cause changes in the levels of skipped product (Figures 4C and 4D). This result implies that exon skipping is not an obligatory consequence of inhibition of the flanking introns by RBM5 and further suggests that PTB and RBM5 are likely to act through distinct molecular mechanisms. Biochemical Mechanism of RBM5-Mediated Splicing Inhibition Previous work indicated that PTB causes Fas exon 6 skipping by interfering with molecular events leading to the stabilization of U2AF by U1 snrnp across the exon (exon definition) (Izquierdo et al., 2005). Several lines of evidence suggest that RBM5 regulates splicing via a different mechanism. First, when the profile of polypeptides crosslinked to exon 6 and neighboring sequences was analyzed in the absence and presence of PTB, the pattern of crosslinked proteins did not change, with the exception of the prominent crosslinking of PTB itself (Izquierdo et al., 2005). In contrast, significant changes in the relative intensity of several crosslinked species were observed upon addition of RBM5 to the extract (Figure 5A). This result suggests that RBM5 causes significant rearrangements in the structure of the ribonucleoprotein particle assembled around Fas exon 6. Despite the various RNA-binding motifs identifiable in RBM5, no direct interaction between the protein and Fas RNA was detected using a variety of experimental approaches, including UV-mediated crosslinking/immunoprecipitation or RNA-protein interaction assays with purified components (data not shown). The possibility that the RNP rearrangement induced by RBM5 leads to decreased association of U2AF with the 3 0 splice site region was analyzed. Crosslinking/immunoprecipitation experiments indicated that RBM5 increased, rather than decreased, U2AF 65 crosslinking to Fas exon splice site, both in the presence and absence of the URE6 silencer or in the absence of the downstream 5 0 splice site (i.e., in the presence or absence of exon definition) (Figure 5B). Repression of U2AF binding by PTB resulted in decreased recruitment of U2 snrnp to the 3 0 splice site region (Izquierdo et al., 2005). In contrast, psoralenmediated crosslinking experiments revealed that neither the association of U2 snrnp nor U1 snrnp with the splice sites flanking exon 6 were affected by RBM5 (Figures 5C and S6). Taken together, these results indicate that splicing repression by RBM5 occurs after the exon becomes defined and prespliceosomal complexes are assembled on the flanking splice sites. Consistent with this notion, prespliceosome (A) complex formation on introns 5 and 6 was not disrupted by addition of RBM5, in contrast with PTB, which caused a decrease in all splicing-related complexes (Figure 5D, lanes 1 4). RBM5 did inhibit formation of fully assembled spliceosomes (complex B), and the decrease in B complex formation was accompanied by higher levels of complex A. These results suggest that RBM5 inhibits the transition of prespliceosomal complexes to spliceosomes. Similar effects were observed for introns 5 and 6, as well as for intron 5, regardless of the presence or absence of a 5 0 splice site downstream of exon 6 (i.e., presence or absence of exon definition) (Figure 5D, lanes 5 12). As expected, in the absence of exon definition, lower levels of complex formation were observed (compare lanes 2 and 6), and PTB did not repress (compare lanes 6 and 8) (Izquierdo et al., 2005), once again arguing that PTB and RBM5 act through distinct molecular mechanisms. RBM5 as a Selector of Splice Site Pairing The results above indicate that RBM5 represses introns 5 and 6 by interfering with a late step in spliceosome assembly. To test whether RBM5 also influences the alternative pathway of splice site pairing involving the 5 0 splice site of exon 5 and the 3 0 splice site of exon 7 leading to exon skipping, minigenes lacking exon 6 (D) An enhancer sequence located in the 3 0 end of exon 6 is important for full RBM5 response. Mutants harboring sequence substitutions of the indicated exonic sequences (Table S3) were analyzed as in (B). (E) The presence of a weak 3 0 splice site upstream of Fas exon 6 is important for response to RBM5. Analysis of the mutants described in (A) and Table S2 was carried out as in (B). Molecular Cell 32, 81 95, October 10, 2008 ª2008 Elsevier Inc. 87

8 Figure 4. RBM5 Inhibits Splicing of Fas Introns 5 and 6 (A) RNA analysis of individual introns in cotransfection assays. RNAs isolated from cotransfection assays as in Figure 3B were analyzed by RT-PCR using either vector-specific primers (PT1-PT2) or combinations of one of these primers and exon 6-specific primers designed to detect splicing of individual introns, as indicated above each panel. 88 Molecular Cell 32, 81 95, October 10, 2008 ª2008 Elsevier Inc.

9 and/or its associated splice sites were generated (Figure 6A). As expected, a single spliced product corresponding to the removal of sequences between exons 5 and 7 was observed (Figure 6B). Depletion of RBM5, 6, and 10 resulted in reduced levels of spliced product accompanied by intron accumulation (Figure 6B, lanes 2 and 5). These effects were reversed upon expression of a sirna-resistant form of RBM5 (Figure 6B, lanes 3 and 6). The results indicate that RBM5 mediates exon 6 skipping not only by repressing introns 5 and 6 but also by displaying an additional activity that promotes distal site utilization. This effect was specific to Fas, because splicing of a comparable minigene derived from the a1-globin genomic region between exons 1 and 3 was not affected in similar experiments (Figure 6C). To understand the role of the distal splice sites and help to further establish the full complement of sequences required for RBM5-mediated control, Fas sequences were engineered into a minigene containing genomic sequences of the a1-globin gene from exons 1 to 3, which is itself barely responsive to RBM5 (Figure 6D, lanes 1 and 2). Note that a cryptic 5 0 splice site within exon 1 is activated in this minigene. Replacement of a-1 globin exon 2 by Fas exon 6 led to constitutive skipping of the exon (lanes 3 and 4, 11 and 12), while replacement of the exon and additional flanking intronic sequences ( 68 to +25) allows efficient inclusion (lane 5). This result suggests that the identity of the flanking splice sites is important for Fas exon 6 definition. The exon, however, was not efficiently regulated by RBM5 (lane 6) unless the distal regions of the a1-globin minigene introns were also replaced by those of the Fas minigene (lanes 7 and 8). The presence of the Fas minigene distal sites did not by itself make the a1-globin minigene RBM5 responsive (lanes 9 and 10). The combination of distal sites and the intronic regions flanking exon 6 (lanes 13 and 14) was not sufficient for RBM5 function either. We conclude that sequences within Fas exon 6 and its flanking splice sites, as well as the distal sites, are necessary for conferring full response to RBM5. Taken together, the results reveal that RBM5 promotes sequence-specific pairing between exon 6-distal splice sites, which leads to exon skipping. An OCRE Domain Required for RBM5 Function Interacts with U5 snrnp Proteins To gain further insights into the mechanism of RBM5 function, we aimed to identify domains of the protein required for Fas splicing regulation. Deletion of the carboxy-terminal region of RBM5 compromised both the association of RBM5 with U2AF complexes (Figure 1D) and the ability of the protein to induce Fas exon 6 skipping (Figure 7A). In contrast, deletion of an OCRE domain (but not of other identifiable domains: Q-rich, coiled coil, zinc finger 2, or G patch within the C-terminal region) (Figure 1E) led to disruption of RBM5 function (Figure 7A and data not shown) but did not compromise significantly the recruitment of the protein to U2AF complexes (Figure 1D, lower panel). These results suggest that the OCRE domain is required for splicing regulation after the factor is targeted to the RNA. Affinity chromatography experiments using an immobilized GST-OCRE fusion protein revealed the specific association of splicing factors (Figures 7B and S7). Consistent with the possibility that RBM5 regulates splice site pairing after exon definition and at the time of U4/5/6 tri-snrnp recruitment, the polypeptides interacting with the critical RBM5 OCRE domain included two components of the U5 snrnp: U5 200K (PRP8) and the helicase U5 220K. Taken together, our results argue that RBM5 regulates alternative splicing in the transition between exon definition-based prespliceosomal assembly and splice site pairing decisions made at the time of tri-snrnp binding, with the U5-contacting OCRE domain playing a key role in this process. DISCUSSION This manuscript reports that the 815 amino acids protein product of the gene RBM5/LUCA-15/H37 is a component of complexes that contain U2AF 65, a key factor in 3 0 splice site recognition. Variations in the levels of RBM5 are shown to modulate alternative splicing of the Fas receptor and the apoptosis regulator c-flip, offering new ground to explain the activities of the RBM5 locus in the control of cell proliferation, programmed cell death, and possibly tumor progression. RBM5 regulates Fas exon 6 by a dual mechanism: (1) it inhibits splicing of the flanking introns without interfering with recognition of exon 6 splice sites by U1 and U2 snrnp but prevents transition from prespliceosomal to spliceosomal complexes and (2) it facilitates the use of the splice sites that become paired during exon skipping. Consistent with regulation occurring in the transition between exon definition and full spliceosomal assembly, an OCRE domain important for RBM5 function interacts with components of the U4/5/6 snrnp. These results can shed new light into the molecular mechanisms of splice site selection. U2AF Complex In addition to confirming known interacting factors like U2AF 35, BBP/SF1, and SPF45, our results suggest that U2AF 65 forms complexes with a significant number of other polypeptides, including proteins containing sequence motifs found in splicing factors that proofread or regulate splice site selection. The presence of most U2 snrnp components is compatible with either spliceosome assembly on pre-mrnas present in the extract (B) RNA analysis of individual introns in RNAi experiments. RNAs isolated as in Figure 2D were analyzed as in (A). The values correspond to average and standard deviation of three independent experiments. (C) RBM5 inhibits splicing of introns 5 and 6 in alternative splicing in vitro assays. In vitro splicing assays were carried out using a pre-mrna containing Fas genomic sequences between exon 5 and 7 (with a neutral internal deletion of intron 6), in the absence or presence of 33 ng/ml of recombinant purified RBM5, and the products of splicing analyzed by primer extension using splice junction primers (Izquierdo et al., 2005). The position of primer extension products corresponding to each alternatively spliced mrna are indicated. Quantification of fold changes in skipping/inclusion ratios (average of the ratios between each signal detecting exon inclusion and the signal corresponding to exon skipping) for three independent experiments are shown. (D) RBM5 immunodepletion or immunoinhibition enhances exon 6 inclusion in vitro. In vitro splicing assays were carried out and analyzed as in (C), using either mock- or RBM5-depleted extracts (left panel) or in the presence of 0.9 mg/ml of control or anti-rbm5 antibodies (right panel). Molecular Cell 32, 81 95, October 10, 2008 ª2008 Elsevier Inc. 89

10 Figure 5. RBM5 Inhibits the Transition between Prespliceosome to Spliceosome Complexes (A) RBM5 induces a rearrangement of interactions between proteins and Fas exon P-radioactively-labeled exon 6 and flanking intronic sequences (from position 68 in intron 5 to +25 in intron 6) was incubated with HeLa nuclear extracts under splicing conditions. After irradiation with UV light, the profile of crosslinked polypeptides was analyzed by electrophoresis on a SDS-polyacrylamide gel. (B) RBM5 enhances U2AF 65 crosslinking to the 3 0 splice site of Fas exon 6. After UV crosslinking as in A, immunoprecipitation of crosslinked U2AF 65 was carried out under conditions that allow specific quantitative detection of changes in U2AF 65 crosslinking to the 3 0 splice site of Fas intron 5 (Izquierdo et al., 2005). Enhancement occurs independently of the presence of the exonic URE6 sequence (m0 mutant) or the downstream 5 0 splice site ( 68 to the 3 0 end of e1, Figure 3A). (C) RBM5 does not inhibit assembly of U1 or U2 snrnp on the splice sites flanking Fas exon 6. Base-pairing interactions between the 5 0 end of U1 snrna and the 5 0 splice site of Fas intron 6 and between U2 snrna and the branch point of intron splice site were analyzed by psoralen-mediated UV crosslinking as described (Izquierdo et al., 2005). Controls for the assignment of crosslinked products are shown in Figure S6. (D) RBM5 inhibits full spliceosome assembly on Fas introns 5 and 6 and causes accumulation of prespliceosomal complex A. Splicing complexes assembled on the indicated Fas RNAs were analyzed by electrophoresis on native gels in the presence or absence of 33 ng/ml of PTB or RBM5, as indicated. The position of hnrnp (H), pre-spliceosome (A), and spliceosome (B) complexes are indicated. or, more likely, with the existence of a U2 snrnp/u2af preassembled complex that may streamline 3 0 splice site recognition. Regulation after Early Splice Site Recognition Targeting initial steps of splice site recognition, e.g., binding of U1 snrnp, U2AF, or the interactions between them that lead to intron or exon definition, are efficient mechanisms of alternative splicing regulation documented in multiple systems (Blencowe, 2006). Recent results, however, indicate that splice site choice decisions can also take place at later steps of the splicing process (reviewed by House and Lynch, 2008). For example, commitment to splicing can be separated from commitment to splice site pairing, allowing kinetic alternative splicing decisions at the step when ATP-driven rearrangements lock U2 snrnp 90 Molecular Cell 32, 81 95, October 10, 2008 ª2008 Elsevier Inc.

11 Figure 6. RBM5 Promotes Sequence-Specific Pairing between the 5 0 Splice Site of Fas Exon 5 and the 3 0 Splice Site of Exon 7 (A) Schematic representation of mutant and chimeric Fas/a1-globin constructs. Exons and introns of each genomic region are represented by different color shades. Crosses indicate mutations that cause splice site inactivation. (B) RBM5 activates splicing of the distal sites. RBM depletion and rescue experiments using Fas minigenes lacking exon 6 or its associated splice sites were carried out as in Figure 2D. The position of the products of splicing between exons 5 and 7, as well as the corresponding intron retention product, are indicated. Molecular mass markers are also shown. (C) A minigene containing a-1 globin genomic sequences is not affected by RBM5, 6, or 10 depletion. Analyses were carried out as in (B). Note that a cryptic 5 0 splice site located 49 nucleotides upstream of exon 1 donor site becomes activated in this minigene. (D) Effects of RBM5 overexpression on a-1 globin and a-1 globin/fas chimeric minigenes. RNA analyses were performed as in Figure 2C using the indicated constructs. The positions of spliced products are indicated. Molecular Cell 32, 81 95, October 10, 2008 ª2008 Elsevier Inc. 91

12 Figure 7. An OCRE Domain Important for RBM5 Function Interacts with Splicing Factors of the U4/5/6 Tri-snRNP (A) An OCRE domain within the C-terminal region of RBM5 is important for Fas alternative splicing regulation. Transfection assays and RNA analyses were carried out as in Figure 2C using wild-type of deletion mutants of RBM5 (and T7-ADAR as control). DC-terminal = deletion of residues ; DOCRE = deletion of residues Values of changes in exon skipping induced by RBM5 are represented as average and standard deviation for three independent experiments. Protein expression was controlled by western blot analyses using anti-t7 epitope antibodies. (B) Pull-down of polypeptides from HeLa nuclear extracts using a GST fusion of the RBM5 OCRE domain and GST as control. The identity of the indicated pulled down polypeptides was determined by mass spectrometry (Figure S7). The asterisk indicates BSA contamination, which was not detected in other pull-down assays. Note that the C terminus of the fusion protein has been extended beyond the defined OCRE motif to facilitate proper folding of this domain, but equivalent results were obtained with a fusion protein including the OCRE domain alone. (C) Model for RBM5-mediated regulation of Fas alternative splicing. RBM5 inhibits splicing of introns 5 and 6 by blocking incorporation of the U4/5/6 tri-snrnp on prespliceosomal complexes assembled in the introns flanking exon 6. In addition, RBM5 promotes pairing between the distal sites, thus acting as a selector of splice site pairing after exon definition. 92 Molecular Cell 32, 81 95, October 10, 2008 ª2008 Elsevier Inc.

13 onto the pre-mrna (Lim and Hertel, 2004). An exonic silencer in the CD45 gene promotes exon skipping after association of U1 and U2 snrnp (House and Lynch, 2006). It is conceivable that particular regulatory signals and factors act through distinct mechanisms on different substrates or depending on the assay system. For example, PTB interferes with early interactions leading to intron or exon definition (Sharma et al., 2005; Izquierdo et al., 2005) but can also act at a step subsequent to U2 snrnp binding when initial splice site recognition has been reinforced (Sharma et al., 2008). The presence of a weak (relatively poor in pyrimidines) 3 0 splice site preceding exon 6 is necessary for efficient RBM5 function. Also important are exonic sequences that include or overlap a splicing enhancer whose function can compensate the weakness of the preceding 3 0 splice site and/or the presence of a PTB-responsive exonic silencer. These observations suggest that RBM5 function depends on the synergistic or antagonistic interplay between regulatory sequences and cognate factors whose balance may be tipped off by RBM5. As RBM5 regulates late events in spliceosome assembly after splice sites have been recognized by U1 and U2 snrnp, it appears that the particular pathway of early splice site recognition (e.g., strong versus enhancer-assisted weak 3 0 splice site and/or the need to overcome PTB-mediated silencing of exon definition) may leave a mark on the assembled complexes that makes them subject to further regulation by RBM5. The additional activity of RBM5 that promotes pairing between the splice sites associated with exons 5 and 7 is also relevant in this context. It is conceivable that RBM5, which is a known component of A complexes that becomes dissociated in the transition to B complexes (Deckert et al., 2006; Behzadnia et al., 2006), acts as a selector of distinct features of U1/U2 complexes assembled on different splice sites to promote incorporation of U4/5/6 particles onto specific pairs of splicing signals while disfavoring others. Given this late coordinating function of partially assembled spliceosomes, it is, perhaps, not surprising that direct interactions between RBM5 and the pre-mrna have not been detected and that an OCRE domain critical for RBM5 function can interact with components of the U4/5/6 tri-snrnp (Figure 7). In vitro biochemical assays dissociate the two activities of RBM5, reproducing inhibition of introns 5 and 6, but not distal site base pairing. As distal site utilization is recapitulated upon inhibition of exon 6 definition by PTB (or upon mutation of exon 6 splice sites), the results imply that distal site utilization may be a default pathway for PTB-mediated, but not RBM5-mediated, regulation. Ibrahim et al. (2005) reported that exonic enhancers bound by SR proteins display a dual function to promote inclusion of internal exons: to enhance the use of the splice sites flanking the exon and inhibit pairing of the distal sites, which leads to exon skipping. RBM5 function requires sequences within exon 6 that coincide or overlap with an exonic enhancer. It is, therefore, conceivable that RBM5 antagonizes the latter function of the enhancer and, thus, relieves its inhibitory effect on distal splice site pairing. Given our limited knowledge of the mechanism of tri-snrnp addition and the rearrangements required to switch from initial exon definition to spliceosome assembly on the flanking introns, deeper understanding of RBM5 function and, in particular, the OCRE domain interactions with U5 snrnp proteins may help to shed light on these important processes. Splicing Regulation of Programmed Cell Death and Tumor Suppression Our results and previous work (Förch et al., 2000; Izquierdo et al., 2005; Corsini et al., 2007) suggest that variations in the levels or activity of RBM5, PTB, SPF45, and TIA-1/TIAR can modulate Fas exon 6 splicing to generate isoforms that activate the extrinsic apoptotic pathway or, alternatively, that sequester the Fas ligand in the extracellular medium and thus inhibit apoptosis (Cheng et al., 1994; Cascino et al., 1995). The existence of various regulatory factors acting through distinct mechanisms can provide a diversity of means by which different cell types or inputs operating under different physiological conditions can modulate programmed cell death. A switch between Fas isoforms occurs, for example, during maturation of T lymphocytes (Liu et al., 1995), and expression of RBM5, although ubiquitous in human tissues, is regulated during thymus development (Drabkin et al., 1999). Altered ratios of Fas isoforms are associated with autoimmune lymphoproliferative syndrome (ALPS) (Roesler et al., 2005) and several other pathologies and can have obvious implications in tumor development and progression. Altered expression of regulatory factors can be at the basis or contribute to the development of some of these pathologies. Indeed, both deletion/ silencing and overexpression of RBM5 have been observed in different classes of tumors. Thus, deletion and silencing of RBM5 is characteristic of lung cancer and is also observed in cancers from other tissues (Sutherland et al., 2005; Maarabouni and Williams, 2006), while activation of the potent Her-2/neu oncogene (observed in 20% 30% of breast and ovarian cancers) leads to upregulation of RBM5 in breast and ovarian cancer cell lines (Oh et al., 2002), and elevated levels of RBM5 have been documented in breast tumor samples (Rintala-Maki et al., 2007). Given the dual function of RBM5 in the control of cell proliferation and apoptosis, genetic alterations in RBM5 expression are likely to contribute to cell transformation, tumor progression, and even to metastasis formation (Ramaswamy et al., 2003). Transcriptional profiling of genes affected by changes in RBM5 expression rendered genes involved in the control of cell cycle and apoptosis (Mourtada-Maarabouni et al., 2006). The function of RBM5 as an alternative splicing regulator reported in this manuscript opens the possibility that changes in splicing contribute to the diversity of functions of this gene in the control of cell growth, programmed cell death, and tumor progression. For example, increased expression of the antiapoptotic c-flip(s) isoform observed upon RBM5 silencing could contribute to lung cancer progression, while decreased expression of the apoptotic form of Fas upon RBM5 overexpression could influence breast cancer progression. The relative contribution of antagonistic factors and isoforms will depend on the rate-limiting steps for transducing apoptotic signals in different cell types or pathological conditions. Cell type-specific cofactors, RBM5 protein modifications, or the balance between the antagonistic functions of RBM5 isoforms or its antisense transcript can all provide additional opportunities for genetic control of the activities of the RBM5 locus and account for the multiple effects of changes in RBM5 expression in cell proliferation, apoptosis, and tumor progression. Molecular Cell 32, 81 95, October 10, 2008 ª2008 Elsevier Inc. 93