Volume 32, Issue 1, Pages 81-95 (October 2008) RBM5/Luca-15/H37 Regulates Fas Alternative Splice Site Pairing after Exon Definition  Sophie Bonnal, Concepción Martínez, Patrik Förch, Angela Bachi, Matthias Wilm, Juan Valcárcel  Molecular Cell  Volume 32, Issue 1, Pages 81-95 (October 2008) DOI: 10.1016/j.molcel.2008.08.008 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 U2AF65/RBM5 Interactions (A) Identification of U2AF65-associated proteins. Immunoprecipitates using anti-U2AF65 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 U2AF65. Precipitates of HeLa nuclear extracts with anti-RBM5 or control antibodies were analyzed by western blot using the MC3 anti-U2AF65 antibody with or without treatment with RNase. (C) Direct interaction of RBM5 and U2AF65. GST-U2AF65 (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 U2AF65 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. ΔC-terminal and ΔOCRE correspond to deletion of residues 321–809 and 452–511 of RBM5, respectively. (E) Arrangement of protein motifs in the primary structure of RBM5. Molecular Cell 2008 32, 81-95DOI: (10.1016/j.molcel.2008.08.008) Copyright © 2008 Elsevier Inc. Terms and Conditions

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′ end of exon 5 and 47 first nucleotides downstream of the 5′ 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 lacking the Q-rich domain (amino acids 362–385), 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 α-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 2008 32, 81-95DOI: (10.1016/j.molcel.2008.08.008) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Exon 6 Sequences and a Weak Associated 3′ 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 6 5′ 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). (D) An enhancer sequence located in the 3′ 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′ 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 2008 32, 81-95DOI: (10.1016/j.molcel.2008.08.008) Copyright © 2008 Elsevier Inc. Terms and Conditions

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. (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/μl 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 μg/μl of control or anti-RBM5 antibodies (right panel). Molecular Cell 2008 32, 81-95DOI: (10.1016/j.molcel.2008.08.008) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 RBM5 Inhibits the Transition between Prespliceosome to Spliceosome Complexes (A) RBM5 induces a rearrangement of interactions between proteins and Fas exon 6. 32P-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 U2AF65 crosslinking to the 3′ splice site of Fas exon 6. After UV crosslinking as in A, immunoprecipitation of crosslinked U2AF65 was carried out under conditions that allow specific quantitative detection of changes in U2AF65 crosslinking to the 3′ 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′ splice site (−68 to the 3′ 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′ end of U1 snRNA and the 5′ splice site of Fas intron 6 and between U2 snRNA and the branch point of intron 5 3′ 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/μl of PTB or RBM5, as indicated. The position of hnRNP (H), pre-spliceosome (A), and spliceosome (B) complexes are indicated. Molecular Cell 2008 32, 81-95DOI: (10.1016/j.molcel.2008.08.008) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 RBM5 Promotes Sequence-Specific Pairing between the 5′ Splice Site of Fas Exon 5 and the 3′ Splice Site of Exon 7 (A) Schematic representation of mutant and chimeric Fas/α1-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 α-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′ splice site located 49 nucleotides upstream of exon 1 donor site becomes activated in this minigene. (D) Effects of RBM5 overexpression on α-1 globin and α-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 2008 32, 81-95DOI: (10.1016/j.molcel.2008.08.008) Copyright © 2008 Elsevier Inc. Terms and Conditions

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). ΔC-terminal = deletion of residues 321–809; ΔOCRE = deletion of residues 452–511. 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. Molecular Cell 2008 32, 81-95DOI: (10.1016/j.molcel.2008.08.008) Copyright © 2008 Elsevier Inc. Terms and Conditions