SRSF3-Regulated RNA Alternative Splicing Promotes Glioblastoma Tumorigenicity by Affecting Multiple Cellular Processes

Abstract

Misregulated alternative RNA splicing (AS) contributes to the tumorigenesis and progression of human cancers, including glioblastoma (GBM). Here, we showed that a major splicing factor, serine and arginine rich splicing factor 3 (SRSF3), was frequently upregulated in clinical glioma specimens and that elevated SRSF3 was associated with tumor progression and a poor prognosis for patients with glioma. In patient-derived glioma stem-like cells (GSC), SRSF3 expression promoted cell proliferation, self-renewal, and tumorigenesis. Transcriptomic profiling identified more than 1,000 SRSF3-affected AS events, with a preference for exon skipping in genes involved with cell mitosis. Motif analysis identified the sequence of CA(G/C/A)CC(C/A) as a potential exonic splicing enhancer for these SRSF3-regulated exons. To evaluate the biological impact of SRSF3-affected AS events, four candidates were selected whose AS correlated with SRSF3 expression in glioma tissues, and their splicing pattern was modified using a CRISPR/Cas9 approach. Two functionally validated AS candidates were further investigated for the mechanisms underlying their isoform-specific functions. Specifically, following knockout of SRSF3, transcription factor ETS variant 1 (ETV1) gene showed exon skipping at exon 7, while nudE neurodevelopment protein 1 (NDE1) gene showed replacement of terminal exon 9 with a mutually exclusive exon 9'. SRSF3-regulated AS of these two genes markedly increased their oncogenic activity in GSCs. Taken together, our data demonstrate that SRSF3 is a key regulator of AS in GBM and that understanding mechanisms of misregulated AS could provide critical insights for developing effective therapeutic strategies against GBMs. SIGNIFICANCE: SRSF3 is a significant regulator of glioma-associated alternative splicing, implicating SRSF3 as an oncogenic factor that contributes to the tumor biology of GBM.

Figure 1.

SRSF3 is upregulated in gliomas and associates with glioma tumor progression and prognosis. A, Heatmap of SR family gene expression in normal brain, LGG, and GBM specimens and statistical analysis of SR gene expression related to tumor grade, IDH1 mutation, and overall survival (OS) of GBM patients based on our RNA-seq data. B, Kaplan-Meier survival analysis for SRSF3 expression in patients with GBM from NU datasets. C, IB analysis for SRSF3 in representative normal brain, LGG and GBM specimens of NU glioma cohort. α/β-tubulin was a loading control. Data are representative of two independent experiments with similar results. D, Quantification of IB data for SRSF3 expression in NU datasets. E and F, Expression of SRSF3 in normal brains and gliomas from TCGA (E) and CGGA datasets (F). G and H, Kaplan-Meier survival analyses for SRSF3 expression. G, TCGA data and H, CGGA data. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance.

Figure 2.

SRSF3 promotes proliferation, self-renewal and in vivo tumorigenicity of GSCs. A and B, Effect of SRSF3-KO in GSC83 and GSC528 (upper panel in A, IB) on cell proliferation (lower panel in A) and sphere-forming frequency (B). C and D, Effect of SRSF3-KO on brain tumor xenograft growth of GSC83 (C) and GSC528 (D) (left, representative BLI images; middle, quantification of BLI), and extended survival of tumor-bearing mice (right, Kaplan Meir analysis). E, IB analysis for expression of endogenous SRSF3 and exogenous Flag-tagged SRSF3 in GSC83 cells with indicated modifications. F to H, Effect of SRSF3 re-expression in SRSF3-KO GSC83 cells on proliferation (F), sphere-forming frequency (G), brain tumor xenograft growth (H) (left, BLI images; middle, quantification of BLI), and survival of tumor-bearing mice (right, Kaplan Meir analysis). Data are representative of two to three independent experiments with similar results. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 3.

Global landscape of SRSF3-affected alternative splicing (AS) in GSCs. A, Quantification of SRSF3-regulated AS events in each category: SE, skipped exons; RI, retained introns; MXE, mutually exclusive exons; A3SS/A5SS, alternative 3’/5’ splice sites. B, Top 10 significantly enriched GO annotations of genes presenting splicing alterations upon SRSF3-KO. C, Changes in PSI values of SRSF3-regulated AS events in each category in indicated GSCs. D, Correlation between RNA Seq-derived ∆PSI and RT-PCR quantified ∆PSI. E, RT-PCR for SRSF3-regulated AS events in five representative genes in GSC83 and 528 cells. ACTB was used as a control. Data are representative of two independent experiments with similar results. F, Flowchart of the SRSF3-motif discovery and functional prediction for SRSF3-dependent exons. G, Isoform-specific function prediction for SRSF3-dependent exons based on ASpedia database.

Figure 4.

Characterization of SRSF3-regulated AS events involved in glioma tumorigenesis. A, Venn diagram of SRSF3-affected AS events identified from SRSF3-KO RNA-seq data and SRSF3-correlated AS events in both TCGA and CGGA glioma datasets. B, RT-PCR validation of four identified SRSF3-regulated AS events. C-F, CRISPR-mediated exon skipping in four candidate genes and their effects on GSC proliferation. P-F/P-R: forward/reverse primers used in RT-PCR; Mut: CRISPR-mediated exon skipping; E: exon. G and H, Effects of exon skipping in ETV1 (G) and NDE1 (H) on sphere-forming frequency of GSCs. I, Exon skipping in ETV1 and NDE1 reduced the growth of GSC83 brain tumor xenografts of nude mice (left, representative BLI images; middle, quantification of BLI) and extended animal survival (right, Kaplan-Meier analysis). Data are representative of two to three independent experiments with similar results. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance.

Figure 5.

Inclusion of exon 7 promotes oncogenic activity of ETV1. A, ETV1-E7-coded region harbors three amino acid residues for MAPK phosphorylation. B, Immunoprecipitation (IP)-IB analyses for exogenous expression of Flag-ETV1 full-length (FL) or -E7-excluded isoform (∆E7). C, RNA-seq analysis. ETV1-target genes were down-regulated upon SRSF3-KO. D, qRT-PCR for ETV1-target genes in GSC83 with SRSF3-KO, ETV1-E7 skipping (ETV1-Mut) or control. E and F, qRT-PCR of ETV1-target genes in GSC83 cells with ETV1-KD or control (E) and in GSC528 cells with ETV1-E7 skipping or control (F). G, RT-PCR (upper panels) and IB (lower panels) analyses of endogenous and exogenous ETV1 expression in GSC83 cells with indicated modifications. H, qRT-PCR of ETV1-target genes in GSC83 cells with indicated modifications. I, Cell proliferation of GSC83 cells with indicated modifications. Data are representative of two to three independent experiments with similar results. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 6.

Isoform-specific function of NDE1 in mitotic spindle formation. A, Schematic of NDE1 protein depicting structural features, binding regions for the indicated NDE1-interacting proteins and alternative last E9. DIC, dynein intermediate chain. DHC, dynein heavy chain. LIS1, Lissencephaly-1. The distinctive amino acids in C-terminus of NDE1-SSSC and NDE1-KMLL isoforms were shown below. B, RT-PCR analysis of endogenous and exogenous NDE1 expression in GSC83 cells with indicated modifications. F: forward primer; R1: reverse primer for NDE1-KMLL isoform; R2: reverse primer for NDE1-SSSC isoform. C, Immunofluorescence (IF) images of DNA (DAPI) and α-tubulin in GSC83 cells with indicated modifications. Scale bar, 5 μm. D, Quantification of the spindle formation defects shown in IF images (C). E, Cell proliferation of GSC83 cells with indicated modifications. F, IB analysis of GSC83 cells that overexpressed GFP-NDE1-KMLL or -SSSC exposed to CHX (20 µg/ml) with indicated time. Right panel, quantification of IB data from three independent experiments. G, IB analysis of GSC83 cells that overexpressed GFP-NDE1-KMLL or -SSSC exposed to CHX (20 µg/ml) with or without MG132 (10 µM) for 12 hr. H, NDE1-E9 splicing pattern in TCGA and CGGA datasets. I, Kaplan-Meier analysis of TCGA and CGGA glioma patients in relation to PSI values of NDE1-E9. Data are representative of two to three independent experiments with similar results. *, p < 0.05; **, p < 0.01; ***, p<0.001.