Nuclear matrix protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB
2015; Springer Nature; Volume: 34; Issue: 5 Linguagem: Inglês
10.15252/embj.201489852
ISSN1460-2075
AutoresMiguel B. Coelho, Jan Attig, Nicolás Bellora, Julian König, Martina Hallegger, Melis Kayikci, Eduardo Eyras, Jernej Ule, Christopher W. J. Smith,
Tópico(s)RNA modifications and cancer
ResumoArticle19 January 2015Open Access Source Data Nuclear matrix protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB Miguel B Coelho Miguel B Coelho Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Jan Attig Jan Attig Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK MRC-Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Nicolás Bellora Nicolás Bellora Computational Genomics, Universitat Pompeu Fabra, Barcelona, Spain Catalan Institute for Research and Advanced Studies (ICREA), Barcelona, Spain INIBIOMA, CONICET-UNComahue, Bariloche, Argentina Search for more papers by this author Julian König Julian König MRC-Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Martina Hallegger Martina Hallegger Department of Biochemistry, University of Cambridge, Cambridge, UK Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK Search for more papers by this author Melis Kayikci Melis Kayikci MRC-Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Eduardo Eyras Eduardo Eyras Computational Genomics, Universitat Pompeu Fabra, Barcelona, Spain Catalan Institute for Research and Advanced Studies (ICREA), Barcelona, Spain Search for more papers by this author Jernej Ule Jernej Ule Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK Search for more papers by this author Christopher WJ Smith Corresponding Author Christopher WJ Smith Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Miguel B Coelho Miguel B Coelho Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Jan Attig Jan Attig Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK MRC-Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Nicolás Bellora Nicolás Bellora Computational Genomics, Universitat Pompeu Fabra, Barcelona, Spain Catalan Institute for Research and Advanced Studies (ICREA), Barcelona, Spain INIBIOMA, CONICET-UNComahue, Bariloche, Argentina Search for more papers by this author Julian König Julian König MRC-Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Martina Hallegger Martina Hallegger Department of Biochemistry, University of Cambridge, Cambridge, UK Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK Search for more papers by this author Melis Kayikci Melis Kayikci MRC-Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Eduardo Eyras Eduardo Eyras Computational Genomics, Universitat Pompeu Fabra, Barcelona, Spain Catalan Institute for Research and Advanced Studies (ICREA), Barcelona, Spain Search for more papers by this author Jernej Ule Jernej Ule Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK Search for more papers by this author Christopher WJ Smith Corresponding Author Christopher WJ Smith Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Author Information Miguel B Coelho1, Jan Attig2,3, Nicolás Bellora4,5,6, Julian König3,7, Martina Hallegger1,2, Melis Kayikci3, Eduardo Eyras4,5, Jernej Ule2 and Christopher WJ Smith 1 1Department of Biochemistry, University of Cambridge, Cambridge, UK 2Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK 3MRC-Laboratory of Molecular Biology, Cambridge, UK 4Computational Genomics, Universitat Pompeu Fabra, Barcelona, Spain 5Catalan Institute for Research and Advanced Studies (ICREA), Barcelona, Spain 6INIBIOMA, CONICET-UNComahue, Bariloche, Argentina 7Present address: Institute of Molecular Biology gGmbH (IMB), Mainz, Germany *Corresponding author. Tel: +44 1223 333655; E-mail: [email protected] The EMBO Journal (2015)34:653-668https://doi.org/10.15252/embj.201489852 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Matrin3 is an RNA- and DNA-binding nuclear matrix protein found to be associated with neural and muscular degenerative diseases. A number of possible functions of Matrin3 have been suggested, but no widespread role in RNA metabolism has yet been clearly demonstrated. We identified Matrin3 by its interaction with the second RRM domain of the splicing regulator PTB. Using a combination of RNAi knockdown, transcriptome profiling and iCLIP, we find that Matrin3 is a regulator of hundreds of alternative splicing events, principally acting as a splicing repressor with only a small proportion of targeted events being co-regulated by PTB. In contrast to other splicing regulators, Matrin3 binds to an extended region within repressed exons and flanking introns with no sharply defined peaks. The identification of this clear molecular function of Matrin3 should help to clarify the molecular pathology of ALS and other diseases caused by mutations of Matrin3. Synopsis Matrin3 is a nuclear matrix protein that was recently linked to neurodegeneration. This study finds Matrin3 to be a splicing repressor that modulates hundreds of alternative splice events, offering possible insight on disease onset. Nuclear matrix protein Matrin3 uses a GILGPPP motif to dock onto the RRM2 domain of splice regulator PTB. Transcriptome-wide profiling shows changes in hundreds of alternative splicing events (ASE) upon Matrin3 knockdown; only a subset of these are also regulated by PTB. Unlike other splicing regulators, Matrin3 binds to extended regions within and around repressed exons. Matrin3 requires its RRMs and the GILGPPP motif to regulate splicing of both PTB-dependent and PTB-independent ASEs, suggesting possible crosstalk with other RRM-containing splice factors. The finding that Matrin3 plays a role in controlling alternative splicing may help understand the etiology of Matrin3-associated pathologies. Introduction Alternative splicing (AS) provides multi-cellular eukaryotes with a proteomic capacity that far exceeds the number of genes (Nilsen & Graveley, 2010). AS is an integral part of regulated programs of gene expression, often acting in concert with transcriptional control, but affecting different functionally related sets of genes (Blencowe, 2006). Regulation of AS is dictated primarily by RNA-binding proteins (RBPs) that can bind to specific RNA sequence elements and which can act as either activators of repressors (Coelho & Smith, 2014). Splicing predominantly occurs co-transcriptionally (Carrillo Oesterreich et al, 2011) in a chromatin context, and this temporal and spatial context provides additional layers of regulatory input into splicing decisions (Braunschweig et al, 2013). Nevertheless, RNA-binding proteins remain the key 'readers' of splicing codes (Barash et al, 2010). RBPs typically have one or more RNA-binding domains, and exhibit varying degrees of specificity, usually recognizing sequence motifs of ~3–5 nt (Ray et al, 2013). While much has been learned about the action of individual RBPs binding to their cognate binding sites, the combinatorial nature of splicing regulation has led to an increased focus on the ways in which groups of regulatory proteins can act together (Barash et al, 2010; Campbell et al, 2012; Zhang et al, 2013; Cereda et al, 2014). Polypyrimidine tract binding (PTB/PTBP1/hnRNPI) protein is an intensively investigated RNA-binding protein, which regulates splicing and other post-transcriptional steps of gene expression (reviewed in Kafasla et al, 2012; Keppetipola et al, 2012; Sawicka et al, 2008). PTB binds to pyrimidine-rich motifs with core CU dinucleotides (Singh et al, 1995; Perez et al, 1997; Ray et al, 2013), and each of its four RRM (RNA recognition motif) family domains (Fig 1A) can recognize such motifs (Oberstrass et al, 2005). Although primarily characterized as a repressive splicing regulator, it can also activate some splice sites and this has been related to differential positions of binding relative to regulated exons (Xue et al, 2009; Llorian et al, 2010). Although PTB can act alone as a regulator (Amir-Ahmady et al, 2005), genome-wide analyses suggest that it cooperates with a number of other proteins as a component of 'tissue spicing codes' (Castle et al, 2008; Wang et al, 2008; Barash et al, 2010; Bland et al, 2010; Llorian et al, 2010). Structure-function analysis has indicated that despite their similar RNA-binding preferences, the four RRMs of PTB show functional diversification (Liu et al, 2002; Robinson & Smith, 2006; Mickleburgh et al, 2014). Of particular importance for synergistic action with other regulators, RRM2 can interact with both RNA via its canonical β-sheet surface, and with short linear PRI (PTB-RRM Interaction) motifs found in the co-regulator Raver1 (Rideau et al, 2006; Joshi et al, 2011). The PRI motif is defined by the consensus sequence [S/G][IL]LGxΦP and binds to the dorsal surface of PTB RRM2, with Tyr247 of PTB particularly critical for this interaction (Rideau et al, 2006; Joshi et al, 2011). PTB RRM2, along with the following linker sequence, is sufficient for splicing repressor activity when artificially tethered as an MS2 fusion protein (Robinson & Smith, 2006) (Fig 1A). Despite the fact that Raver1 can act with PTB as a co-regulator of Tpm1 splicing (Gromak et al, 2003; Rideau et al, 2006), Raver1 null mice showed no alteration in Tpm1 splicing (Lahmann et al, 2008) and knockdown of Raver1 in HeLa cells showed only a few changes in alternative splicing (Hallegger et al, manuscript in preparation). Therefore, it remains possible that other co-regulatory proteins with PRI motifs might interact with PTB RRM2. Figure 1. PTB RRM2 interacts with multiple RNA-binding proteins Schematic representation of PTB (top) and the GST-PTB RRM2 (below), with the limits of the PTB minimal repressor domain indicated. PTB is composed of four RNA recognition motifs (RRM) with three linker regions in between them. It also contains a N-terminus bipartite nuclear localization signal (NLS) as well as a nuclear export signal (NES). The GST-PTB RRM2 is composed of the second RRM fused to a GST tag in the N-terminus. Silverstain of the GST-PTB RRM2 pull-down of the wild-type RRM2 and the Y247Q mutant. Five microlitre of the pull-down was run on a 15% SDS–PAGE and silver-stained. Three strong bands can be seen which are due to the recombinant protein and the beads used for the pull-down. The region encompassing 50 kDa to the top of the gel was sliced and subjected to in-gel digestion and mass spectrometry. The two strongest bands visible in this region are labelled as Matrin3 and Raver1. Proteins identified in the GST-PTB RRM2 pull-down ranked by their unique peptide number. The table shows the different proteins we found binding to RRM2, as well as when present, the sequence of the PRI motif. The indicated function is only a guideline as many have more functions than shown. Western blot of the GST pull-down using antibodies against Matrin3 and Raver1. Lanes 1 and 2 show 5 and 10% of input, respectively, lanes 3 and 4 show GST-PTB full-length pull-down of wild-type and Y247Q mutant, respectively, and lanes 5 and 6 show GST-PTB RRM2 pull-down of wild-type and Y247Q mutant, respectively, and lane 7 with pull-down using GST alone. Source data are available online for this figure Source Data for Figure 1 [embj201489852-sup-0007-source_data_Fig1.jpg] Download figure Download PowerPoint Matrin3 is one of the most abundant inner nuclear matrix proteins (Nakayasu & Berezney, 1991). The main isoforms of Matrin3 are over 800 amino acids in size, but most of the protein is not comprised of structurally characterized domains, with the exception of two DNA-binding C2H2 zinc finger (ZF) and two RRM domains (Hibino et al, 2006), and a bi-partite nuclear localization signal (NLS) (Hisada-Ishii et al, 2007) (Fig 2A). Matrin3 is essential for viability of some cells (Hisada-Ishii et al, 2007; Przygodzka et al, 2010), and alterations in Matrin3 levels are associated with some diseases (Bernert et al, 2002; Bimpaki et al, 2009). Moreover, missense mutations in Matrin3 have been associated with asymmetric myopathy with vocal cord paralysis (Senderek et al, 2009) and amyotrophic lateral sclerosis (ALS) (Johnson et al, 2014). Matrin3 is located diffusely throughout the nucleoplasm and is concentrated in the nuclear scaffold (Zeitz et al, 2009), and its DNA- and RNA-binding domains suggest that it may play roles in processes associated with the nuclear matrix or nucleoplasm. It can anchor chromosomes to the nucleus matrix by binding to the MAR/SAR elements (Hibino et al, 1992). Introduction of MAR/SAR sites upstream of a promoter stimulates transcription, suggesting Matrin3 binding to these elements might promote transcription (Hibino et al, 2000), a suggestion supported by the proximity of Matrin3 with RNA Pol II promoters (Malyavantham et al, 2008) and enhancers (Skowronska-Krawczyk et al, 2014). Matrin has also been shown to be involved in the early stages of the DNA double-strand break response (Salton et al, 2010). A number of functional roles associated with cellular and viral RNA have been suggested for Matrin3 including mRNA stabilization (Salton et al, 2011), nuclear retention of hyperedited RNA (Zhang & Carmichael, 2001) and Rev-dependent export of unspliced HIV1 RNA in conjunction with PTB-associated factor (PSF) (Kula et al; Kula et al, 2011; Yedavalli & Jeang, 2011). Matrin3 interacts with a number of splicing regulators including hnRNPK (Salton et al, 2011), hnRNPL, SFRS7, p68 (Zeitz et al, 2009), NOVA-1/-2 (Polydorides et al, 2000), CTCF (Fujita & Fujii, 2011; Shukla et al, 2011), as well as the transcription machinery itself (Das et al, 2007). Despite the interactions with splicing factors, there is no direct evidence for Matrin3 functioning as a splicing regulator. Figure 2. Matrin3 interacts with PTB via a PRI motif Schematic representation of Matrin3. Matrin3 is composed by two zinc finger (ZF) domains, two tandem RNA recognition motifs (RRM), as well as a N-terminal nuclear export signal (NES) and a C-terminal nuclear localization signal (NLS). A PRI motif is localized between the first ZF and the first RRM, and the sequence is aligned with the sequence from the two functional PRIs from Raver1. Conserved PRI residues are in bold. FLAG immunoprecipitation of Matrin3 and Raver1, both with wild-type and with PRI mutated, and FLAG-MS2 as a negative control. The immunoprecipitated complex was separated in a SDS–PAGE and subjected to Western blot using antibody against PTB which showed interaction to wild-type Matrin3 (lane 2) and Raver1 (lane 4). The input was also analysed by Western blot with antibodies against PTB as a loading control and against the FLAG tag to ensure equal expression of proteins. The 20-residue Raver1 491–511 (lane 1) and the Matrin3 346–365 (lane 3) peptide fused to MS2 were transcribed and translated in vitro (Input) and then pulled down with GST-PTB or with GST-SXL as a control. Effects of single mutation of the Matrin3 PRI GILPPP to GIAPPP were also tested (lane 4). Source data are available online for this figure Source Data for Figure 2 [embj201489852-sup-0008-source_data_Fig2.jpg] Download figure Download PowerPoint Here, we set out to identify nuclear proteins that interact with PTB RRM2. Matrin3 was the major interacting protein in HeLa nuclear extracts, interacting via a single PRI motif that is necessary and sufficient for interaction. Using RNAi knockdown and splice-sensitive microarray analysis in conjunction with iCLIP of Matrin3 and PTB, we find that Matrin3 acts widely as a splicing regulator. While a number of its target splicing events are shared with PTB, the majority are PTB independent and involve Matrin3 action as a repressor. Matrin3 binding was observed in the introns flanking repressed exons, but in contrast with other splicing regulators, the binding occurred to an extended region with no clear peaks. Structure-function analysis indicates that Matrin3 splicing activity requires both the RRM domains and the PRI, even for ASEs that are not co-regulated by PTB. Results Identification of PTB RRM2 binding partners With the aim of understanding better the function of the minimal PTB repressor domain, we carried out a proteomic screen to identify interacting protein partners of PTB RRM2, the main component of the repressor domain (Robinson & Smith, 2006). PTB RRM2 was fused to GST in wild-type (WT) and Y247Q mutant form, which impairs interaction with Raver1 PRI peptides (Joshi et al, 2011) (Fig 1A), and used as bait to pull down interacting proteins from HeLa nuclear extracts. Numerous proteins bound to WT RRM2 but not the Y247Q mutant (Fig 1B). Proteins pulled down by WT GST-RRM2 were identified by mass spectrometry (Fig 1C and Supplementary Table S1). They include RNA-binding proteins (MATR3, RAVER1, HNRNPM, RBMX, DDX5, DDX3X, SFRS15, DDX17, HNRNPH1 and PTB itself), proteins with role in transcription regulation (CCAR1, KIAA1967/CCAR2 and RUVBL1/2) and a protein found in 3′ end processing complexes (WDR33). Four of the five unique PTB peptides are located within RRM2 and so could derive from the bait protein. Proteins with roles in transcription and 3′ end processing may present links to unknown activities of PTB in the case of transcription regulation, and in the case of WDR33 (Shi et al, 2009), a molecular link to an already reported function of PTB in 3′ end processing (Moreira et al, 1998; Castelo-Branco et al, 2004). The strongest protein interaction detected, as indicated by number of unique peptides and MASCOT score, was the nuclear matrix protein Matrin3, followed by Raver1 (Fig 1C and D). These two proteins correspond to the major protein bands interacting specifically with WT but not mutant RRM2 (Fig 1B, arrows). Matrin3, CCAR1, KIAA1967 and WDR33 all have potential PRI motifs similar to those in Raver1 (Figs 1C and 2A). We validated the Matrin3-PTB interaction by Western blot of GST-RRM2 and full-length GST-PTB pull-downs, comparing wild-type (WT) and Y247Q mutant proteins (Fig 1D). Both Matrin3 and Raver1 interacted strongly with GST-RRM2 and GST-PTB proteins, and in both cases, the Y247Q mutation abolished the interaction. This indicates that the RRM2 interaction is sufficient and also necessary in the context of full-length GST-PTB for interaction with Matrin3 and Raver1 (Fig 1D). However, while Matrin3 interacted equally well with RRM2 or full-length PTB, Raver1 interacted more strongly with full-length PTB, suggesting that other regions of PTB may also contact Raver1. Matrin3 PRI motif is necessary and sufficient for PTB interaction Matrin3 is a large nuclear protein with 847 amino acids that can bind both to DNA via two C2H2 zinc finger domains (ZF1 and ZF2) and to RNA by its tandem RNA recognition motifs (RRM1 and RRM2) (Hibino et al, 2006). A potential PRI motif, GILGPPP, is located between ZF1 and RRM1. This matches the PRI consensus (Fig 2A) and is located in a disordered region, which is important for the function of short linear motifs (Dinkel et al, 2014). Moreover, the motif is absolutely conserved across 84 mammalian, avian, reptilian and amphibian species (UCSC browser, Vertebrate Multiz Alignment & Conservation, 100 Species). In order to test whether the GILGPPP motif is functional, we mutated it to GAAAPPA (mutated residues underlined) in a FLAG-tagged Matrin3 expression vector and tested the effect on PTB binding by anti-FLAG co-immunoprecipitation. As control, we used wild-type Raver1 and a mutant with all four PRI motifs mutated (Rideau et al, 2006). FLAG-tagged Matrin3 and Raver1 both co-immunoprecipitated PTB (Fig 2B). Mutation of the single PRI in Matrin3 nearly eliminated PTB co-immunoprecipitation (lane 3), a more emphatic effect than mutation of the Raver1 PRI motifs (Fig 2B, lane 5). We next tested whether the Matrin3 PRI is sufficient for binding to PTB. We in vitro transcribed and translated the Matrin3 and the Raver1_1 PRIs (Fig 2A) fused to the bacteriophage MS2 coat protein. Both the Raver1 and Matrin3 peptides were pulled down by GST-PTB (Fig 2C, lanes 1, 3). As negative controls, no binding was observed to an unrelated RNA-binding protein, GST-SXL, and MS2 alone was not pulled down by GST-PTB or GST-SXL (lane 2). The specificity of the interaction was demonstrated by mutation to alanine of the conserved leucine-3 of the PRI, which strongly impaired binding to PTB (Fig 2C, lane 4). These data therefore demonstrate that the PRI motif of Matrin3 is both necessary and sufficient for interaction with PTB. Matrin3 is a widespread regulator of alternative splicing The interaction of Matrin3 with PTB led us to hypothesize that it may play a role in the co-regulation of some PTB-regulated alternative splicing events (ASEs). To test this hypothesis, we transfected HeLa cells with siRNAs targeting the Matrin3 mRNA and observed a > 90% decrease in the Matrin3 protein levels (Fig 3A). Total RNA from knockdown and control samples was purified and analysed using Human Junction microarrays (HJAY), containing probe sets for all annotated human exons and exon–exon junctions (Llorian et al, 2010). The array data were analysed using the ASPIRE3 pipeline. Only 61 genes showed changes in RNA levels of greater than twofold, including the expected reduction of Matrin3 levels (3.7-fold; Supplementary Table S2). This suggests, in contrast to a previous report (Salton et al, 2011), that Matrin3 does not play a widespread role in stabilizing mRNAs. We did observe down-regulation of some of the previously reported mRNAs (Salton et al, 2011), but also observed alteration of alternative splicing events towards isoforms of these mRNAs with premature termination codons, which normally leads to nonsense-mediated decay (see 3). Figure 3. Global splicing effects upon Matrin3 knockdown Western blot probed for Matrin3 (top panel), PTB (middle panel) and actin (lower panel). Lanes 1–4 contain a twofold dilution of the control C2 sample (lane 1—12.5%, lane 2—25%, lane 3—50%, lane 4—100%). Lanes 4–7 contain equal amount of proteins, as can be confirmed by the anti-actin (lower panel), of control sample (lane 4), double-PTB and nPTB siRNA-treated sample (lane 5), Matrin3 siRNA-treated sample (lane 6) and triple knockdown of Matrin3, PTB and nPTB (lane 7). Lane 7 is from the same gel and exposure, but some lanes present in the original gel were cropped for clarity, and a black line to indicate cropping was placed. Pie chart of the different categories of Matrin3-regulated alternative splicing events (ASEs), 330 cassette exons (50%), 116 alternative promoter usage (17%), 78 terminal exons (11%), 32 alternative 5′ (5%) and 34 3′ (5%) splice site and 18 intron retention (IR; 3%) events. Gene ontology (GO) analysis of the Matrin3-regulated cassette exons. The x-axis represents the P-value in a logarithmic scale as shown. Pie chart of the activated and repressed cassette exons by Matrin3. Venn diagram of the overlap between the PTB- (blue) and Matrin3- (yellow) regulated cassette exons, showing the 813 events regulated only by PTB, 270 by Matrin3 only and the 61 that overlap. RT–PCR validation of Matrin3-regulated alternative spicing events in the ST7, ACSL3, PLEKHA3, TCF12, VWA5A, PTBP2, PTBP3, C3orf17, ZMYND8, VEZT, PIGX and DMD genes. In each case, triplicates for each condition (C—control, M—Matrin3, PTB/nPTB and Matrin3/PTB/nPTB siRNA transfection samples) were analysed and exon inclusion (EI) percentage is shown beneath the corresponding lane, along with the standard deviation (s.d.). Source data are available online for this figure Source Data for Figure 3 [embj201489852-sup-0009-source_data_Fig3.jpg] Download figure Download PowerPoint Next, we examined the potential role of Matrin3 in regulating alternative splicing. Significant changes in splicing were predicted by ASPIRE using a threshold of |dIrank| > 1 (Supplementary Table S3), which has previously been shown to produce a validation rate of > 80% (König et al, 2010; Wang et al, 2010). This identified 667 ASEs, half of which were cassette exons (n = 331; 50%; Fig 3B). Of the Matrin3-regulated cassette exons, 75% showed increased inclusion upon Matrin3 knockdown, indicating that Matrin3 represses inclusion of these exons (Fig 3D). Notably, the degree of confidence in the changes observed in splicing of the 25% Matrin3-activated cassette exons was lower when compared to the Matrin3-repressed ones (Supplementary Fig S1). We next examined the cassette exons that may be jointly regulated by Matrin3 and PTB, using the HJAY data set produced upon knockdown of PTB and PTBP2 (Llorian et al, 2010). The double knockdown is essential as upon PTB knockdown, its neuronal paralogue PTBP2 is upregulated and can partially compensate for loss of PTB (Boutz et al, 2007; Makeyev et al, 2007; Spellman et al, 2007). Only 61 (18.4%) of the 331 Matrin3-regulated cassette exons were also regulated by PTB (Fig 3E). While the number of co-regulated cassette exons is 2.2-fold greater than expected by chance (expected 27.4, P = 5.5e−10, hypergeometric test), the majority of the Matrin3-regulated ASEs are PTB independent. We validated a number of the cassette exon events predicted to be regulated by Matrin3 and PTB by knockdown of Matrin3 or PTBP1/PTBP2. We also tested the effects of combined knockdown of Matrin3/PTBP1/PTBP2 (Fig 3A). RT–PCR was carried out using primers in flanking constitutive exons and the percentage exon inclusion determined (Fig 3F). Four different classes of events were observed, depending on their response to Matrin3 and PTB knockdown: Matrin3 repressed, PTB independent (ST7 exon 11, ACSL3 exon 3 and PLEKHA3 exon 4); Matrin3 activated, PTB repressed (TCF12 exon 18, VWA5A exon 2, PTBP2 exon 10 and PTBP3 exon 2); Matrin3 repressed, PTB activated (C3orf17 exon 2, ZMYND8 exon 33 and VEZT exon 11); and repressed by both Matrin3 and PTB (PIGX exon 3 and DMD exon 78). In the cases where Matrin3 and PTB activities were opposed, knockdown of the repressor protein had a larger effect and tended to be dominant over the activator. In some cases, knockdown of the activator had no effect in the absence of the repressor (e.g. VEZT exon 11 and PTBP2 exon 10), suggesting that that the sole function of the activator is to antagonize the repressor. Properties of Matrin3-regulated exons In order to assess whether the exons regulated by Matrin3 possess any specific splicing features, we examined 5′ and 3′ splice sites, branch points, pyrimidine tracts, and nucleotide composition (Supplementary Fig S3) and flanking intron lengths (Fig 4A). Few significant differences were observed compared to a control set of annotated cassette exons unaffected by knockdown of Matrin3 (or PTB) knockdown. One striking difference was that the introns flanking Matrin3 repressed exons are on average 1 kb longer than introns flanking Matrin3-activated, PTB-repressed, PTB-activated or control exons (Fig 4A). Figure 4. Bioinformatic analysis of Matrin3-regulated splicing events Intron lengths flanking exons regulated by Matrin3, PTB or control exons. Kruskal–Wallis rank-sum test was used to test for significant changes (Matr3 P-value = 3.947e−15, PTB P-value = 0.3896). ***P < 0.001. Diagram of Matrin3-repressed cassette exons with enriched RBP motifs for human proteins (Ray et al, 2013) shown above and the number of enriched pentamers shown below in each of 7 locations. The RBP motifs are shown with their consensus binding site logo (Ray et al, 2013) and the respective motif enrichment score (odds ratio). The significant enriched k-mers are shown in Supplementary Table S4, and all enriched RBP motifs (multiple species) are shown in Supplementary Table S5. Download figure Download PowerPoint We next looked for enrichment of pentamer sequence motifs associated with Matrin3-regulated exons, compared to control unregulated cassette exons, across seven transcript locations (cassette exons, flanking constitutive exons, 5′ and 3′ end of each flanking intron). Numerous motifs were enriched (FDR < 0.05) in the introns flanking Matrin3-repressed exons, but none within exons or in any location associated with Matrin3-activated exons (Fig 4B, Supplementary Fig S4, Supplementary Table S4). Motifs associated with Matrin3-repressed exons were heterogeneous and included a number of pyrimidine motifs associated with PTB (e.g. TTCTT, TCTTT). The enrichment was also observed using a control set consisting of exons including PTB-regulated, Matrin3-independent exons. Most of the remaining motifs had high pyrimidine content, with one or two interrupting purines; more than half of the motifs immediately flanking Matrin3 repressed exons had a single purine. Individual analyses of RNA binding by Matrin3 have not revealed a clear consensus sequence (Hibino et al, 2006; Salton et al, 2011; Yamazaki et al, 2014). However, Matrin3 was one of 207 RBPs whose optimal sequence was determined by the RNA-compete array-based selection (Ray et al, 2013). We therefore used heptamer position frequency matrices to look for enrichment of RNA-compete motifs. Once again, enriched motifs were found only in the introns flanking Matrin3-repressed exons, and in no locations associated with Mat
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