Splicing factor hnRNPH drives an oncogenic splicing switch in gliomas
2011; Springer Nature; Volume: 30; Issue: 19 Linguagem: Inglês
10.1038/emboj.2011.259
ISSN1460-2075
AutoresClare V. LeFave, Massimo Squatrito, Sandra Vorlová, Gina Rocco, Cameron Brennan, Eric C. Holland, Ying‐Xian Pan, Luca Cartegni,
Tópico(s)RNA regulation and disease
ResumoArticle13 September 2011Open Access Splicing factor hnRNPH drives an oncogenic splicing switch in gliomas Clare V LeFave Clare V LeFave Department of Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Weill Graduate School of Medical Sciences, Pharmacology Program, New York, NY, USA Search for more papers by this author Massimo Squatrito Massimo Squatrito Brain Tumor Center, MSKCC, New York, NY, USA Department of Cancer Biology and Genetics, MSKCC, New York, NY, USA Search for more papers by this author Sandra Vorlova Sandra Vorlova Department of Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Search for more papers by this author Gina L Rocco Gina L Rocco Department of Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Weill Graduate School of Medical Sciences, Pharmacology Program, New York, NY, USA Search for more papers by this author Cameron W Brennan Cameron W Brennan Brain Tumor Center, MSKCC, New York, NY, USA Department of Neurosurgery, MSKCC, New York, NY, USA Search for more papers by this author Eric C Holland Eric C Holland Brain Tumor Center, MSKCC, New York, NY, USA Department of Cancer Biology and Genetics, MSKCC, New York, NY, USA Department of Neurosurgery, MSKCC, New York, NY, USA Search for more papers by this author Ying-Xian Pan Ying-Xian Pan Department of Neurology, MSKCC, New York, NY, USA Search for more papers by this author Luca Cartegni Corresponding Author Luca Cartegni Department of Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Brain Tumor Center, MSKCC, New York, NY, USA Experimental Therapeutics Center, MSKCC, New York, NY, USA Search for more papers by this author Clare V LeFave Clare V LeFave Department of Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Weill Graduate School of Medical Sciences, Pharmacology Program, New York, NY, USA Search for more papers by this author Massimo Squatrito Massimo Squatrito Brain Tumor Center, MSKCC, New York, NY, USA Department of Cancer Biology and Genetics, MSKCC, New York, NY, USA Search for more papers by this author Sandra Vorlova Sandra Vorlova Department of Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Search for more papers by this author Gina L Rocco Gina L Rocco Department of Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Weill Graduate School of Medical Sciences, Pharmacology Program, New York, NY, USA Search for more papers by this author Cameron W Brennan Cameron W Brennan Brain Tumor Center, MSKCC, New York, NY, USA Department of Neurosurgery, MSKCC, New York, NY, USA Search for more papers by this author Eric C Holland Eric C Holland Brain Tumor Center, MSKCC, New York, NY, USA Department of Cancer Biology and Genetics, MSKCC, New York, NY, USA Department of Neurosurgery, MSKCC, New York, NY, USA Search for more papers by this author Ying-Xian Pan Ying-Xian Pan Department of Neurology, MSKCC, New York, NY, USA Search for more papers by this author Luca Cartegni Corresponding Author Luca Cartegni Department of Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Brain Tumor Center, MSKCC, New York, NY, USA Experimental Therapeutics Center, MSKCC, New York, NY, USA Search for more papers by this author Author Information Clare V LeFave1,2, Massimo Squatrito3,4, Sandra Vorlova1, Gina L Rocco1,2, Cameron W Brennan3,5, Eric C Holland3,4,5, Ying-Xian Pan6 and Luca Cartegni 1,3,7 1Department of Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York, NY, USA 2Weill Graduate School of Medical Sciences, Pharmacology Program, New York, NY, USA 3Brain Tumor Center, MSKCC, New York, NY, USA 4Department of Cancer Biology and Genetics, MSKCC, New York, NY, USA 5Department of Neurosurgery, MSKCC, New York, NY, USA 6Department of Neurology, MSKCC, New York, NY, USA 7Experimental Therapeutics Center, MSKCC, New York, NY, USA *Corresponding author. Department of Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY, USA. Tel.: +1 646 888 2168; Fax: +1 646 422 0271; E-mail: [email protected] The EMBO Journal (2011)30:4084-4097https://doi.org/10.1038/emboj.2011.259 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 In tumours, aberrant splicing generates variants that contribute to multiple aspects of tumour establishment, progression and maintenance. We show that in glioblastoma multiforme (GBM) specimens, death-domain adaptor protein Insuloma-Glucagonoma protein 20 (IG20) is consistently aberrantly spliced to generate an antagonist, anti-apoptotic isoform (MAP-kinase activating death domain protein, MADD), which effectively redirects TNF-α/TRAIL-induced death signalling to promote survival and proliferation instead of triggering apoptosis. Splicing factor hnRNPH, which is upregulated in gliomas, controls this splicing event and similarly mediates switching to a ligand-independent, constitutively active Recepteur d′Origine Nantais (RON) tyrosine kinase receptor variant that promotes migration and invasion. The increased cell death and the reduced invasiveness caused by hnRNPH ablation can be rescued by the targeted downregulation of IG20/MADD exon 16- or RON exon 11-containing variants, respectively, using isoform-specific knockdown or splicing redirection approaches. Thus, hnRNPH activity appears to be involved in the pathogenesis and progression of malignant gliomas as the centre of a splicing oncogenic switch, which might reflect reactivation of stem cell patterns and mediates multiple key aspects of aggressive tumour behaviour, including evasion from apoptosis and invasiveness. Introduction In mammalian genes, exons must be properly spliced together to generate mature mRNAs (Cartegni et al, 2002). Failure to effectively and accurately utilize splice sites can generate unstable and/or aberrant mRNAs encoding for defective or deleterious protein isoforms (Faustino and Cooper, 2003). Furthermore, splice sites can be differentially selected depending on developmental state, tissue and cell type, or in response to a wide array of physiological and pathological signals. Alternative splicing (AS) affects >75% of mammalian genes and is responsible for much of the proteome complexity (Pan et al, 2008). AS regulation ultimately depends on the intrinsic strength of the splice sites, on the presence of cis-regulatory elements (intronic and exonic enhancers and silencers), and on the combinatorial control by a discrete number of trans-activating factors, typically belonging to the serine/arginine-rich splicing factor (SR proteins) or the heterogeneous nuclear ribonucleoprotein (hnRNP) families (Martinez-Contreras et al, 2007; Long and Caceres, 2009). A growing body of evidence suggests that dysregulated splicing patterns are also associated with tumourigenesis, with the appearance of hundreds of cancer-related isoforms. Multiple AS transcripts have been identified as progression markers, indicating both generalized splicing abnormalities or tumour- and stage-specific events (Venables et al, 2009). Malignant transformation is dependent on the acquisition of specific capabilities, from uncontrolled growth, to escape from apoptosis, to metastatic invasion (Hanahan and Weinberg, 2000), typically obtained through mutations or genomic rearrangements. The same oncogenic effects can result from various epigenetic mechanisms, including the modification of AS patterns. In tumours, aberrant splicing usually arises from variations in the relative amounts/activity of regulatory splicing factors. Although a definitive causal relationship remains to be established for most cases, it is clear that the expression of specific splice variants of many cancer-related genes can directly contribute to the oncogenic phenotype and has a determinative role in many aspects of tumourigenesis and in the development of resistance to treatments (Ghigna et al, 2008). Glioblastoma multiforme (GBM) is the most common type of primary brain cancer and is associated with a dismal prognosis, primarily due to its infiltrating properties and the emergence of resistance (Kanu et al, 2009). Treatment options have remained limited in part because of the still incomplete understanding of the basic biology of GBM. Given the complexity of splicing regulation in the brain, aberrant splicing in gliomas may be a significant but yet under-explored contributor to the heterogeneous pathological characteristics of GBM. Indeed, glioma-specific AS patterns (Cheung et al, 2008) have been reported, as well as additional aberrant splicing events that can contribute to all aspects of gliomagenesis, affecting angiogenesis (Huang et al, 2005), oncogenic suppression (Chunduru et al, 2002), escape from apoptosis (Yamada et al, 2003), proliferation (Camacho-Vanegas et al, 2007; Yu et al, 2007), metabolism (Clower et al, 2010; David et al, 2010) and migration/invasion (Yu et al, 2007; Cheung et al, 2009; Lo et al, 2009). To better understand the role of AS deregulation in GBM, we looked at previously described splicing events that are altered in a variety of cancers and can generate variants with experimentally verified oncogenic properties. Here, we report that, in a large majority of human GBM samples and corresponding mouse models, exon 16 of the death-domain Insuloma-Glucagonoma protein 20 (IG20) transcript is differentially spliced to express the survival isoforms MAPK-activating death-domain-containing protein (MADD; Al-Zoubi et al, 2001). IG20/MADD, an adaptor protein involved in TNF-α and TRAIL signalling (Mulherkar et al, 2007; Kurada et al, 2009), triggers apoptosis through caspase 8 activation (Al-Zoubi et al, 2001; Mulherkar et al, 2007). Alternative 5′ splice site usage in exon 13 combined with the inclusion/skipping of exon 16 generates four main splicing isoforms (Figure 1A), including MADD/DENN variants. While IG20 triggers apoptosis through caspase 8 activation, MADD variants are necessary and sufficient for cell survival in vitro and in vivo and their knockdown enhances TRAIL-induced apoptosis in cancer cells (Al-Zoubi et al, 2001; Lim et al, 2004; Mulherkar et al, 2006, 2007). MADD variants have been described to be aberrantly expressed in tumours (Efimova et al, 2004; Prabhakar et al, 2008; Kurada et al, 2009) and may thus constitute an important component of tumour escape mechanisms. Figure 1.IG20/MADD exon 16 alternative splicing is altered in gliomas. (A) Schematic of the exon structure of four IG20/MADD isoforms generated by AS of exon 13 (alternative 5'ss) and exon 16 (exclusion). RT–PCR using primers flanking exons 13 and 16 (black arrows) shows the splicing pattern of the four isoforms in HeLa cells. Arrows indicate approximate position of primer pairs. (B) Total RNAs from human normal brain (n=5) and GBM samples (n=20) were analysed by RT–PCR for IG20/MADD exon 16 splicing pattern using primer sets on exons 14 and 17 (red arrows in A). Representative gels are shown. (C) Quantification of data from (B) for AS of IG20/MADD exon 16 (left), IG20/MADD exon 13L (middle) and positive control FGFR1 α-exon (right). Three PCRs were quantified and averaged for each sample. The 90/10/median box and whiskers plot was then calculated for the normal (n=5) and tumour (n=20) sets using Prism software. The 90/10/median shows the variation of exon inclusion of the calculated normal and tumour sets. Indicated P-values were determined by two-tailed Student's t-test. (D) IG20/MADD exon 16 splicing pattern (as in B) from the indicated human tissues and cell lines. (E) Three independent mouse brain samples of the N-tva Ink-4a-Arf−/− LoxP PTEN background were examined for AS of the murine IG20/MADD exon 16 pattern along five independent samples each from brain tumour developed in the same genetic background following RCAS-PDGF and RCAS-PDGF+RCAS-CRE delivery. In short, all samples are Ink-4a-Arf null and tumours are driven by PDGF-B overexpression alone or concomitant to PTEN downregulation. RT–PCR experiments using murine IG20 primers in exons 14 and 17 were repeated in triplicate, and a representative gel is shown with the average quantification of the exon 16 inclusion is below. The drops from inclusion levels in the normal brain (green columns) to the levels in the two tumour groups (orange and yellow columns) are highly statistically significant (pvals=1.57E-16 and 4.27E-08, respectively). Download figure Download PowerPoint Splicing factors are often overexpressed in tumours and can directly behave as potent proto-oncogenes. SRSF1 (previously called SF2/ASF; Manley and Krainer, 2010) is upregulated in various human cancers and its overexpression is sufficient to transform rodent fibroblasts and cause high-grade sarcomas in nude mice (Karni et al, 2007). SRSF1 also directly modulates the expression of tumourigenic Recepteur d′Origine Nantais (RON) isoforms (Ghigna et al, 2005). RON is the tyrosine kinase receptor for the macrophage stimulating protein (MSP), and is highly homologous to mesenchymal–epithelial transition receptor, whose activation in GBM is associated with shorter survival and poor prognosis (Kong et al, 2009). RON is a heterodimeric transmembrane receptor involved in cell proliferation, survival and the promotion of the epithelial–mesenchymal transition (EMT) and invasion (Lu et al, 2007). Exon 11 exclusion generates an isoform, RONΔ11 (RON165) that lacks part of the extracellular domain, resulting in a constitutive active isoform that promotes cell motility and mediates EMT (Ghigna et al, 2005). In the present study, we describe that two oncogenic exonic silencing events (IG20 exon 16 and RON exon 11) occur in GBM samples and both can be controlled by the AS factor hnRNPH, which is overexpressed in gliomas. Control of these two splicing silencing events by hnRNPH can occur through an identical exonic splicing silencer (ESS) located at the 5′ end of the skipped exons, suggesting the same mechanism of action. Our data suggest a novel role for hnRNPH as a splicing regulator in GBM biology, which can contribute to multiple pathological aspects of the GBM phenotype. Results AS of IG20/MADD exon 16 is altered in human and mouse gliomas To study IG20/MADD AS in gliomas (McLendon et al, 2008; Lo et al, 2009), total RNAs from 20 GBM and 5 non-tumour brain samples were analysed by semi-quantitative reverse transcriptase PCR (RT–PCR). A representative result set for IG20/MADD exon 16 is shown in Figure 1B and a quantification of multiple experiments in Figure 1C, along with control PCRs. In non-tumour brain, we observed consistent levels of above 40% exon 16 inclusion (42.12±1.442 s.e.m., N=5), whereas inclusion of exon 16 in our 20 GBM samples dropped to a mean value of ∼15% (15.08±3.640 s.e.m., N=20, pval=5.11E-07; Figure 1C). This sharp decrease in exon 16 inclusion level was observed in 95% of the patient samples (19/20). On the contrary (Figure 1C; Supplementary Figure S1), no changes were observed when the AS pattern of IG20/MADD exon 13 was analysed (39.98±0.9 versus 41.40±2.1), suggesting that the two events are independently regulated and that switching to the tumourigenic MADD variant is specifically modulated in gliomas. As a positive control, we analysed FGFR1 exon-α splicing, a well-characterized AS event occurring in gliomas (Yamaguchi et al, 1994), which was similarly included less in GBM than in non-tumour brain (41.20±4.620 versus 78.20±1.428, pval=1.27E-07; Figure 1C; Supplementary Figure S1). Extension of the analysis of IG20/MADD AS to a panel of normal tissues and cell lines (Figure 1D) shows that exon 16 inclusion varies broadly in various tissues, from mostly skipped (e.g., thyroid) to mostly included (skeletal muscle), suggesting that this is a highly regulated and tissue-specific event. Furthermore, transformed cell lines show a more marked exon 16 skipping when compared with non-transformed ones (Figure 1D, right panel). To investigate IG20/MADD exon 16 splicing in glioma mouse models, we took advantage of the RCAS/tva system, in which the RCAS avian leukosis virus mediates somatic gene transfer into cells expressing the viral receptor (tva) (Dai and Holland, 2001). Localized delivery of RCAS retroviruses expressing the PDGF-B gene into the brain of newborn transgenic mice producing the tva receptor under control of the early glial lineage nestin promoter (N-Tva) gives rise to glial tumours (Dai and Holland, 2001; Shih et al, 2004). When PDGF-B is expressed in N-Tva mice with Ink-4a-Arf null background (N-Tva Ink-4a-Arf−/−) or with conditional PTEN knockout background (N-Tva LP, together with RCAS-Cre), tumour initiation and progression is promoted, yielding higher grade gliomas (Hu and Holland, 2005). These GBM-like features are enhanced by the combination of both backgrounds (N-Tva Ink-4a-Arf−/− LPTEN; Hambardzumyan et al, 2009). Similarly to what observed in human GBM samples, we determined that RNAs derived from the PDGF-driven high-grade tumours showed a consistent switch towards IG20/MADD exon 16 skipping when compared with RNAs from control brains of the same genotype, where PDGF-B was not expressed (Figure 1E, compare lanes 1–3 with lanes 4–8 or 9–13 and quantification below). IG20/MADD exon 16 contains multiple regulatory elements The mechanism(s) controlling IG20/MADD AS are currently unknown. To identify regulatory cis-elements that modulate exon 16 splicing and the corresponding trans-acting factors, we generated a minigene construct containing the 3061-nt genomic region spanning IG20/MADD exons 15–17, with the entire intervening introns (Figure 2A). Transient expression of this synthetic pre-mRNA yielded a splicing pattern comparable to the endogenous transcripts (∼10% inclusion, Figure 2B, lanes 1 and 2), indicating that most relevant regulatory elements are maintained in the minigene. Strengthening of the weak pyrimidine tract of exon 16 by a double mutation (Figure 2A, Py^), resulted in full exon 16 inclusion (Figure 2B, lane3), suggesting that its incorporation depends on efficient recognition of the splice sites. Figure 2.Identification of regulatory exonic splicing elements in IG20/MADD exon 16. (A) Diagram of AS of IG20/MADD minigene (top) with the sizes (nt) indicated. Wild-type (wt) and pyrimidine enhanced (Py^) partial minigene sequences are shown (lowercase and uppercase letters represent introns and exons, respectively). Lines above and below the exon 16 sequence indicate exact position of the 12-nt (Δ1–Δ5, above) and 3-nt (1a–5c, below) deletions generated. Red and green boxes represent the approximate mappings of the identified putative ESS and of the three putative ESEs. (B) Splicing pattern of endogenous IG20/MADD exon 16 (endo) and transfected wt and Py^ minigenes (exo) in HeLa cells. For the endogenous products, primers on exons 14 and 17 are used, for the minigene product, plasmid-specific primers are used. (C) RT–PCR analysis of exon 16 deletion mutants within the wt-minigene context, transfected into HeLa cells. The positions of the deletions on exon 16 are indicated. Download figure Download PowerPoint Then, we used these minigenes as the basis for a systematic mutational analysis of exon 16. A first set of partially overlapping deletions (Δ1–Δ5) was introduced from position +3 to +55 (Figure 2A). Deletion Δ1 lead to a dramatic switch to full exon 16 inclusion (Figure 2C, lane 1) identifying this region as a putative ESS. Smaller deletions encompassing Δ1 narrow the putative silencer to the TTTGGG sequence from position 3 to 9 of the exon (Figure 2C, lanes 7 and 8). Deletions Δ2–Δ5 are associated with a reduction in exon 16 inclusion (Figure 2C, lanes 2–5), suggesting the presence of exonic splicing enhancers (ESEs). To better study these elements, we inserted the same deletions in the context of the Py^ mutant, where the basal level is full inclusion, and confirmed that ablation of any of the Δ2–Δ5 regions inhibits exon 16 inclusion (Supplementary Figure S2A). Smaller deletions analysed in the same context revealed the presence of three distinct ESEs in exon 16 (Supplementary Figure S2B). A bioinformatics analysis of the sequence using enhancer-predicting algorithms identified three clusters of putative enhancer elements that colocalize with the regions identified by deletion analysis (Supplementary Figure S2C). However, overexpression of putative regulating splicing factors SRSF1, SRSF5 and SRSF6 leads to no changes in exon 16 splicing (Supplementary Figure S2D). A comparable mutational analysis using larger deletions (100/500 nt) spanning most of the upstream intron did not yield any candidate intronic regulatory element (Supplementary Figure S3). IG20/MADD exon 16 silencer is controlled by hnRNPH To characterize the silencer revealed by the TTT and GGG deletion, we analysed by transient transfection a complete panel of mutants where every position was independently changed to all three other nucleotides (Figure 3A and B). Figure 3.The ESS in IG20/MADD exon 16 is controlled by hnRNPH. (A) RT–PCR analysis of mutant minigenes harbouring single-point mutations generated within deletions 1a and b (Figure 2A and C) upon transient transfection in HeLa cells. Top indicates the wt nucleotide and the position within IG20/MADD exon 16. Left-down indicates the nucleotide each position is mutated to. *Indicates that the wild-type nucleotide was maintained. Representative gels of three independent transfections experiments are shown. (B) Quantification of point mutations made in (A) represented as percent of exon 16 skipping; data are average of three independent experiments, ±s.d. (C) Representation of a pseudo-frequency matrix obtained from data in (B), generated using WebLogo 3.0. (D) EMSA of radiolabelled wt-probe mock treated (lanes 1 and 9), or incubated with HeLa nuclear extract (lanes 2 and 10), with × 20, × 100 or × 400 excess of unlabelled wild-type (wt) or mutant (mut) probes (lanes 3–5 and 6–8, respectively), with a control IgG (lane 11) or with hnRNPH-specific antibody N-16 (lane 12). Supershifted band is indicated by arrow. RNA sequences for the wt and mut probes are shown on top with the putative hnRNPH binding site region underlined, and mutation in red. (E) Wt IG20/MADD minigene and G7–A7 mutant (Figure 2A, lane 5 panel 'A') co-transfected with control siRNAs (siC) or with siRNAs to HnRNPH (siH1). RT–PCR of exogenous IG20/MADD exon 16 splicing (top), western blots for total hnRNPH (middle) and actin (bottom). (F) HeLa cells were separately treated with two individual siRNAs to hnRNPH, twice 24 h apart and then RNAs were collected at 72 h for analysis. Top, RT–PCR analysis of endogenous MADD exon 16 splicing. Bottom, two panels are western blot analyses of hnRNPH and actin. Download figure Download PowerPoint When any of the Gs from the G-triplet (G6–G8 in the exon) were mutated, exon 16 skipping dropped from ∼90% to <25%. On the contrary, mutation of the second T of the element (T4 in the exon) has no effect on the splicing pattern, whereas mutation of the first T (T3 in the exon) leads to high levels of inclusion only if changed to G (Figure 3A and B, lane 1 panel G). Since the preceding nucleotide in the exon is an A, this mutant introduces an AG just three nucleotides downstream of the natural AG. Sequencing of the corresponding PCR product revealed in fact that this additional AG is efficiently used as an alternative 3′ splice site (Supplementary Figure S4). Mutation of the third T (T5 in the exon) to A or C strongly inhibits exon 16 skipping, while on the contrary transversion to a G promotes even stronger exon skipping (Figure 3A and B, lane 3 panel G), indicating that the T is important in maintaining the silencing activity, but it can be substituted by a G. A pseudo-frequency matrix of the four nucleotides in the six different positions was derived from the exon 16 inclusion quantitation data and the WebLogo 3.0 algorithm (Crooks et al, 2004) was used to generate a consensus motif pictogram (Figure 2C). The consensus sequence T/G GGG corresponds to the well-characterized binding motif of hnRNPH/F proteins (Chen et al, 1999; Caputi and Zahler, 2001). HnRNPH is an RNA-binding protein involved in multiple aspects of RNA metabolism. The splicing activity of hnRNPH is highly context dependent and can both inhibit or promote usage of specific splice sites from either intronic or exonic positions (Fogel and McNally, 2000; Mauger et al, 2008; Fisette et al, 2010). A radioactively labelled 24 nt RNA probe spanning the putative hnRNPH binding site, incubated with nuclear extract, formed a slow-migrating complex, identified by electrophoretic mobility shift assay (EMSA; Figure 3D, lane 2). This complex is competed away by excess amount of unlabelled RNA of the same sequence (lanes 4 and 5), but not by equal amounts of a mutant RNA comprising the single G-to-A mutation within the UGGG motif (Figure 3D, lanes 7 and 8), previously shown to induce exon 16 inclusion (Figure 3A, lane 5). Inclusion of antibodies specific to hnRNPH, but not of control antibodies, induced a supershift in the migration profile, demonstrating that the complex formed around the UGGG motif includes hnRNPH (Figure 3D, lanes 11 and 12). SiRNA-mediated knockdown of hnRNPH leads to a net increase in exon 16 inclusion from the wild-type minigene, but not from the G-to-A mutant (Figure 3E). Endogenous IG20/MADD splicing was also similarly affected when either of two hnRNPH-targeted siRNAs was used (Figure 3F). Attempts to overexpress hnRNPH were not very successful, perhaps because of the presence of negative feedback mechanisms to regulate its levels (Ni et al, 2007). However, co-transfection of a plasmid encoding hnRNPH with the IG20 minigene was associated with a further decrease in exon 16 inclusion in the wt, but not in the mutant construct (Supplementary Figure S5) even if the levels of hnRNPH were not obviously elevated. Based on these observations and on abundant data that show that hnRNPH can act directly through such motifs in regulating splicing, we conclude that hnRNPH limits exon 16 inclusion by binding to the UGGG-containing silencer at the 5′ end of exon 16. HnRNPH protects from cell death via MADD activity Since MADD promotes cell survival, whereas IG20 promotes apoptosis, the switch from MADD to IG20 driven by hnRNPH depletion should result in reduced cell viability. Indeed, compared with control siRNA treatments, knockdown of hnRNPH by siH1/2 (Figure 4A, lanes 2 and 5), led to a significant increase in cell death both in U373 glioma cells and in HeLa cells (U373: 2.2±0.20-fold increase, pval=0.00012; HeLa: 3.9±0.97-fold increase, pval=6.43E-05; Figure 4C) that remarkably parallels the improved exon 16 inclusion (quantified in Figure 4B). Figure 4.HnRNPH regulates IG20-dependent cell death through IG20/MADD splicing. (A) U373 glioma cells (left) and HeLa cells (right) were treated for 72 h with the indicated siRNAs (siH1/2=siH1+siH2). HnRNPH knockdown was assessed by western blot using actin as a loading control. The pattern of IG20/MADD exon 16 splicing was analysed by gel electrophoresis and a representative gel is shown on the bottom. (B) Exon 16 inclusion was quantified from multiple biological replicates (U373, n=6; HeLa, n=7) and is represented as average percent of exon 16 inclusion (±s.d.). (C) Cell death from the experiments in (B) was determined by trypan blue assay and is represented as fold change of control treatment (±s.d.). P-values were calculated by two-tailed Student's t-test. Download figure Download PowerPoint HnRNPH controls a broad number of targets, many of which are still unknown. To test whether the increase in cell death is directly caused by the change in IG20/MADD splicing, rather than by other unrelated hnRNPH-dependent events, we combined knockdown of hnRNPH with isoform-specific knockdown of IG20, using an siRNA targeted to exon 16 (siE16). When siE16 was combined with the siRNAs targeting hnRNPH (siH1/2), downregulation of the IG20 isoform led back to IG20/MADD RNA ratios similar to those of control-related cells (Figure 4A and B, lanes 1 versus 3 and 4 versus 6), regardless of hnRNPH levels (top western). This switchback in the splicing profile is associated with robust rescue from hnRNPH-induced cell death in both cell types (Figure 4C, lanes 3 and 6), indicating that hnRNPH not only significantly promotes cell survival, but also that its action is specifically mediated by inhibiting inclusion of IG20 exon 16. RON exon 11 splicing is also controlled by hnRNPH When we examined other AS variants differentially expressed in tumours and suggested to have a role in various aspects of tumourigenesis, we found that RON exon 11 was also significantly more excluded in GBM samples than in non-tumour samples (Figure 5A and B, 44.07±3.324 s.e.m. versus 60.03±4.981, pval=0.0258), although the change was not as homogeneous as in the case of IG20/MADD. We were unable to reliably detect the mRON transcripts by RT–PCR in mouse samples, but when we examined the splicing pattern of RON in human normal tissues and cell lines (Supplementary Figure S6) levels of exon 11 exclusion were overall lower in the tissues
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