Fig. 6: BUD31 promotes BCL2L12 exon 3 inclusion through direct binding to the pre-mRNA. | Nature Communications

Fig. 6: BUD31 promotes BCL2L12 exon 3 inclusion through direct binding to the pre-mRNA.

From: Splicing factor BUD31 promotes ovarian cancer progression through sustaining the expression of anti-apoptotic BCL2L12

Fig. 6: BUD31 promotes BCL2L12 exon 3 inclusion through direct binding to the pre-mRNA.The alternative text for this image may have been generated using AI.

a The AS pattern and BUD31 direct binding sites in BCL2L12 were visualized with IGV using RNA-seq, RIP-seq, and SpyCLIP-seq data. The light blue region highlights the alternative exon and the BUD31-binding sites. b The Sashimi plots of exon 3 skipping in BCL2L12 in HEYA8 cells with BUD31 knockdown (red) and corresponding controls (blue). c, d Semi-quantitative RT-PCR and fragment analysis were performed to validate AS events in BCL2L12 after BUD31 knockdown or overexpression. e The relative expression ratio of BCL2L12-L/BCL2L12-S was analyzed in HEYA8 ovarian cancer cells with BUD31 knockdown or overexpression (n = 3 biologically independent experiments). f Schematic structure of two BCL2L12 transcripts. BCL2L12-L is the full-length transcript, and BCL2L12-S is a short transcript lacking exon 3 skipping, which generates a premature termination codon (PTC). g BCL2L12-S expression was measured by qPCR in UPF1 knockdown and control HEYA8 cells treated with 10 μg/ml actinomycin D at the indicated times. h RIP-qPCR was performed to validate the interaction between BUD31 and BCL2L12 RNA in HEYA8 cells with the anti-BUD31 antibody (n = 3 biologically independent experiments). *p < 0.05, **p < 0.01. i RNA pull-down assay showed the interaction between BCL2L12 pre-mRNA and BUD31 protein. Protein expression was quantified and normalized with the Input sample. j RNA EMSA showed the binding of recombinant BUD31 and BCL2L12 pre-mRNA fragments. The upper band shows the complex of BUD31 protein and BCL2L12 pre-mRNA. k Relative BCL2L12-L/S transcript expression was measured by qPCR in SOC samples (n = 8) and FTs (n = 4). l Isoform percentage of BCL2L12-L and BCL2L12-S in SOCs and normal ovaries from the TCGA-OV and GTEx datasets. m Correlation between BUD31 mRNA expression and the PSI value of AS events in the TCGA-OV dataset. P values and r values were calculated by Pearson’s correlation. n Kaplan–Meier analyzes the correlation between BCL2L12 exon 3 expression and overall survival based on TCGA data. The p values were obtained by two-tailed unpaired Student’s t-test (e, g, h) or log-rank test (n), and the results are presented as the mean ± SD. Source data are provided as a Source Data file.

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