Splicing factor hnRNPH drives an oncogenic splicing switch in gliomas

Abstract

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.

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).

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.

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.

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.

Figure 5

A similar ESS in RON exon 11 is also controlled by hnRNPH. (A) Total RNAs from normal brain (n=5) and GBM samples (n=20) were analysed by RT–PCR for AS of RON exon 11 using primer sets on exons 10 and 13. The difference in size is due to exon 11 skipping. Representative gels are shown. (B) Quantification of data from (A) for AS of RON exon 11, as in Figure 1C. (C) Alignment of the 5′ regions of RON exon 11 and IG20 exon 16 with the TGGG motifs underlined (indicated by red boxes). (D) RT–PCR analysis of endogenous RON exon 11 splicing pattern from samples treated with siRNAs to hnRNPH (siH1) for up to 72 h (top). Western blot analysis of hnRNPH expression levels (middle) from the same treatments, with actin as loading control (bottom). (E) Schematic of AS of RON exon 11 minigene with exon and intron sizes and the SRSF1 binding site indicated (top). ^*Indicates the position of the RON minigene A and B G-to-A mutations within the TGGG motif, where m11a is 5′ to m11b and m11ab is a combination of the two. (F) Wt RON minigene and m11ab double mutant (from ) co-transfected with control siRNAs (siC) or with siRNAs to hnRNPH (siH1). RT–PCR of exogenous RON exon 11 splicing (top), western blots for total hnRNPH (middle) and actin (bottom).

Figure 6

FSD-NMD knockdown of hnRNPH reduces invasiveness via RON exon 11 splicing. (A) Left: FSD-NMD knockdown of hnRNPH. Morpholinos targeted to hnRNPH exon 4 splice sites (H4.3′ and H4.5′) cause skipping of exon 4, leading to a frameshift and a PTC in exon 5, and ultimately causing RNA degradation by NMD. Right: splicing redirection. Morpholinos targeted to RON exon 11 splice sites (R11.3′ and R11.5′) induce in-frame skipping of exon 11, resulting in two protein products: ligand-dependent full-length RON and ligand-independent RONΔ11. (B) Knockdown of hnRNPH by FSD-NMD in HeLa cells after 72 h of treatment (control, H4.3′, H4.5′ and combined H4.3′+H4.5′). The abundance of hnRNPH RNAs following FSD-NMD morpholino treatment was quantified by qPCR and represented as percent downregulation of control treatment (average of two independent experiments). Bottom panels: western blot analysis of hnRNPH protein levels, with actin as loading control. (C) T98G glioma cells and HeLa cells were treated with control morpholinos (lanes 1 and 4), with the H4 pair of hnRNPH knockdown morpholinos (lanes 2 and 5) and with the H4 pair combined with R11 pair of RON splicing redirection morpholinos (lanes 3 and 6). The effect of treatments on RON exon 11 splicing pattern (RT–PCR), and on hnRNPH or actin levels (western blots) was analysed. The same cells were also analysed for their invading capabilities using a matrigel invasion assay (bottom panel). Invading cells were stained with crystal violet and scored blind. Values were then normalized to the control for each experiment. Averages of normalized scores from 15 (5 experiments in triplicate) and 9 (3 experiments in triplicate, ±s.d.) matrigel inserts are represented for T98G and HeLa cells, respectively. P-values were determined by a two-tailed Student's t-test.

Figure 7

HnRNPH is overexpressed in gliomas. (A) qPCR analysis of hnRNPH expression in normal (n=5) and GBM (n=20) RNAs. Results were normalized to the housekeeping gene rps3 and are represented as −dCt. (B) HnRNPH expression from five microarray studies. Oncomine™ (Compendia Bioscience, Ann Arbor, MI) was used for analysis and visualization. Bredel et al: brain (1), GBM (2). Shai et al: white matter (1), GBM (2). Sun et al: brain (1), GBM (2). Liang et al: brain (1), cerebellum (2), GBM (3). Lee et al: neural stem cells (1), GBM (2). Expressed as normalized expression units. (C) Immunohistochemistry analysis of human normal brain, low-grade glioma and high-grade glioma (GBM) samples using anti-H antibodies. Scale bar indicates 100 μm. For a larger panel of samples analysed, see Supplementary Figure S10. (D) Western blot analysis of samples from Figure 1E, using anti-hnRNPH antibodies, with tubulin shown as loading control.