The aberrant splicing of BAF45d links splicing regulation and transcription in glioblastoma

Abstract

Background: Glioblastoma, the most aggressive primary brain tumor, is genetically heterogeneous. Alternative splicing (AS) plays a key role in numerous pathologies, including cancer. The objectives of our study were to determine whether aberrant AS could play a role in the malignant phenotype of glioma and to understand the mechanism underlying its aberrant regulation.

Methods: We obtained surgical samples from patients with glioblastoma who underwent 5-aminolevulinic fluorescence-guided surgery. Biopsies were taken from the tumor center as well as from adjacent normal-appearing tissue. We used a global splicing array to identify candidate genes aberrantly spliced in these glioblastoma samples. Mechanistic and functional studies were performed to elucidate the role of our top candidate splice variant, BAF45d, in glioblastoma.

Results: BAF45d is part of the switch/sucrose nonfermentable complex and plays a key role in the development of the CNS. The BAF45d/6A isoform is present in 85% of over 200 glioma samples that have been analyzed and contributes to the malignant glioma phenotype through the maintenance of an undifferentiated cellular state. We demonstrate that BAF45d splicing is mediated by polypyrimidine tract-binding protein 1 (PTBP1) and that BAF45d regulates PTBP1, uncovering a reciprocal interplay between RNA splicing regulation and transcription.

Conclusions: Our data indicate that AS is a mechanism that contributes to the malignant phenotype of glioblastoma. Understanding the consequences of this biological process will uncover new therapeutic targets for this devastating disease.

Fig. 1

Fluorescence-guided surgery (FGS) and glioblastoma alternative splicing (AS) candidate discovery. (A) Image of 5-ALA FGS during glioblastoma resection. a: Image of the surgical field using a white-light module; b: image of the surgical field under 5-ALA fluorescence, where red/pink and blue correspond to the tumor and macroscopically normal tissue, respectively. (B) Schematic representation and representative agarose PCR gels of the AS of the following candidates obtained from global splicing analysis: BAF45d, RALGAPA, ITAG6, ARHGEF7, BBX, LRRFIP2, and NDE1. Normal brain (N); tumor (T). (C) Validation of the 7 candidate genes using conventional PCR in 10 additional patient samples. Quantification of the Percent Splicing Index (PSI) provides the inclusion level of each exon. Error bars represent the mean ± SD.

Fig. 2

Characterization of BAF45d alternative splicing in glioblastoma. (A) Left: A schematic representation of the BAF45d exon structure, showing that exon 6a is present in BAF45d/6A+ (in normal and normal-appearing tissue) but not in BAF45d/6A− (in the tumor). Lower right: Representative photograph of a conventional PCR gel showing the presence of the 2 isoforms (6A+ and 6A−) in the normal brain and the single isoform (6A−) in the tumor. Upper right: Quantification of the Percent Splicing Index (PSI) provides the inclusion level of each exon. (B) BAF45d protein isoform structures obtained from ensembl (www.ensembl.org). Inclusion of exon 6a in the BAF45d/6A+ isoform disrupts the protein domains formed by exons 6 and 7. (C) mRNA expression of BAF45d in normal tissue (n = 9), normal-appearing tissue (normal-like [LK], n = 20), and glioblastoma (tumor, n = 50) samples. (D) Expression of BAF45d/6A− and BAF45d/6A+ mRNA represented as the ratio (BAF45d/6A−/BAF45d/6A+) in glioblastoma, normal-appearing, and normal tissue samples. (E) BAF45d expression heatmap generated from The Cancer Genome Atlas RNA-Seq data corresponding to 166 glioblastomas and 116 oligodendrogliomas.

Fig. 3

BAF45d/6A− contributes to the tumorigenicity and maintenance of an undifferentiated phenotype in glioma cell lines. (A and B) The proliferation index quantified by MTS in GSC23 cell lines upon (A) inhibition of the BAF45d/6A− isoform (siBAF45d/6A−), the whole gene (siBAF45d), or the control siScramble (siScrbl); (B) overexpression of the empty vector pcDNA-3.1 (pcDNA), pcDNA-3.1_6A+ concomitant with the inhibition of pLKO1_scramble (pcDNA_6A+/pLKO_Scrbl), or pCDNA-3.1_6A+ concomitant with the inhibition of pLKO1_BAF45d/6A− (pcDNA_6A+/pLKO_6A-). (C) Kaplan–Meier graph of the overall survival of mice bearing orthotopic cells depleted of BAF45d (shBAF45d) compared with shScramble mice (shScbrl). (D) Kaplan–Meier graph of the overall survival of mice bearing orthotopic cells with stable overexpression of BAF45d/6A+ (pcDNA_6A−) compared with mice with empty pcDNA-3.1 (pcDNA). (E) Left: The Ki67 index, which represents the percentage of the total labeled nuclei in 10 high-power fields (40x), in the control group (shScrbl) and the BAF45d-inhibited group (shBAF45d). Right: Microscopic (20x) Ki67 immunohistochemical images corresponding to the control group (shScrbl) and the BAF45d-inhibited group (shBAF45d). (F) Left: Ki67 index quantified in the control group (pcDNA) and the group overexpressing the BAF45d/6A+ isoform (pcDNA_6A+). Right: Microscopic (20x) Ki67 immunohistochemical images corresponding to the control group (shScrbl) and the BAF45d-inhibited group (shBAF45d). (G) Self-renewal capacity assay in GSC23 cells transfected with a scramble siRNA (siScrbl) or siBAF45d/6A−. (H) Self-renewal capacity assay in GSC23 cells transfected with an empty pcDNA-3.1 (pcDNA) or overexpressing BAF45d/6A+ concomitant with transfection with pLKO1 carrying a scramble short hairpin (sh) (pcDNA_6A+/pLKO_Scrbl) or overexpressing BAF45d/6A+ concomitant with inhibition of the BAF45d/6A− isoform with an sh specific for that isoform (pcDNA_6A+/pLKO_6A−). (I) Representative western blot of markers associated with an undifferentiated cell phenotype after the indicated treatments in GSC23. Representative PCR image of the different BAF45d isoforms after transfection with an empty pcDNA-3.1 (pcDNA) or overexpressing BAF45d/6A+ concomitant with transfection with pLKO1 carrying a scramble sh (pcDNA_6A+/pLKO_Scrbl) or overexpressing BAF45d/6A+ concomitant with inhibition of the BAF45d/6A− isoform with an sh specific for that isoform (pcDNA_6A+/pLKO_6A−). Quantification of the Percent Splicing Index (PSI) is reported as the mean ± SD of 3 independent experiments. (J) Forced differentiation of GSC23 in 10% fetal bovine serum medium for 10 days. Sex determining region Y–box 2 representative western blot. Representative PCR image of the different BAF45d isoforms after forced differentiation. Quantification of the PSI is reported as the mean ± SD. (K) BAF45d/6A− and BAF45d/6A+ mRNA expression in SH-SY5Y neuroblastoma cells instructed with staurosporin to differentiate toward neurons or stably transfected with pcDNA_6A+/pLKO_6A− as control. In all panels, error bars represent the mean ± SD.

Fig. 4

PTBP1 mediates exon 6a exclusion in BAF45d pre-RNA. (A) Cross-linking immunoprecipitation (CLIP) of PTBP1-bound BAF45d RNA. Reverse transcription quantitative (RT-q)PCR identified the BAF45d region bound by PTBP1 in vivo. The locations of primer pairs (OXF/R) along the BAF45d transcript are indicated in the diagram above. BS: number of binding sites. Glyceraldehyde 3-phosphate dehydrogenase was used as a negative control. *P = 0.01 compared with an immunoglobulin G control. (B) Left: Expression of mRNA of BAF45d/6A−, BAF45d/6A+, and PTBP1 in U87MG cells after inhibition of PTBP1 (siPTBP1) compared with expression with scramble control (siScrbl). RNA expression was quantified by RT-qPCR. Lower right: Representative photograph of a conventional PCR gel showing the expression of BAF45d/6A− and BAF45d/6A+ isoforms after siPTBP1 or siScramble (siScrbl). Upper right: Quantification of the Percent Splicing Index (PSI) provides the inclusion level of each exon. (C) Upper panel: Representative western blot depicting the inhibition of PTBP1. Lower panel: Proliferation index quantified by MTS in the U87MG cell line upon inhibition of PTBP1 (siPTBP1) or the control siScramble (siScrbl). (D) A proposed model for exon 6a splicing of BAF45d by PTBP1. The diagram shows the exon 6a sequence (underlined) and part of the flanking introns. Green bases correspond to the sequence of the 3 regions (branch point, 3ʹ, and 5ʹ splice sites) that are initially recognized by the splicing machinery (spliceosome). SnRNP proteins that specifically recognize these regions are included. Depicted in red are the binding motifs for specific recognition by PTBP1. PTBP1 is represented in its monomeric form with 4 RNA recognition motif (RRM) domains. In the model, the binding of PTBP1 leads to the formation of an mRNA exon loop, and then exclusion of the exon from the spliced mRNA. In all panels, error bars represent the mean ± SD.

Fig. 5

BAF45d positively regulates PTBP1 and splicing events. (A) Assessment of PTBP1 mRNA (upper panel) and protein expression (lower panel) after siBAF45d/6A− or SiScramble (siScrbl) in the GSC23 cell line. (B) Assessment of PTBP1 mRNA (upper panel) and protein expression (lower panel) after pcDNA (control empty vector), pcDNA_BAF45d/6A+/pLKO_Scrbl, or pcDNA_6A+/pLKO_6A− in the GSC23 cell line. (C) Enrichment (relative to input) of BAF45d in the GSC23 PTBP1 promoter as determined by ChIP-qPCR. (D) Enrichment (relative to input) of BAF45d in the GSC23 PTBP1 promoter after transfection with pcDNA_BAF45d/6A+/pLKO_Scrbl or pcDNA_6A+/pLKO_6A− in the GSC23 cell line as determined by ChIP-qPCR. (E) Schematic representation and representative pictures of agarose PCR gels depicting the inversion in the splicing pattern of genes regulated by PTBP1 after treatment with siBAF45d/6A− or siScramble (siScrbl). (F) Scheme depicting a proposed model for the role and mechanism of action of BAF45d during gliomagenesis and neurogenesis. In all panels, error bars represent the mean ± SD.