Fig. 4: RBFOX2 expression is required for the SON knockdown-mediated PTBP2 exon 10 inclusion, and RBFOX2 and SON compete with each other for binding to the target RNA.

a RT-qPCR analyses of PTBP1 and PTBP2 expression in U87MG cells after knockdown of SON and/or RBFOX2 as indicated (n = 3). b The protein levels of SON, PTBP1, PTBP2, and RBFOX2 after knockdown of SON and/or RBFOX2 were analyzed by Western blotting. Actin was used as a loading control. WB Data are representative of n = 3 independent experiments. c RT-PCR analyses of PTBP2 exon 10 inclusion upon SON knockdown and/or RBFOX2 knockdown. PBX1 alternative splicing was shown as a control that is not regulated by RBFOX2. Data are representative of n = 3 independent experiments. d CLIP-PCR assay demonstrating the direct interaction between the SON protein and PTBP2 RNA (exon 9 to 11 region). Data are representative of n = 3 independent experiments. e A diagram of the human PTBP2 pre-mRNA with four RBFOX2-binding motifs (yellow boxes) and the locations of the primer sets (color-coded arrows designated with the numbers 1–8) that were used for the CLIP-qPCR assays shown in (f–h). f SON-binding to PTBP2 pre-mRNA was measured by CLIP with a SON antibody followed by qPCR using the primer sets indicated by the numbers 1–8 (n = 3). Control IgG was used for a CLIP control. The bar graph shows qPCR signals amplified from the CLIP assays as percentage of amplification of the input RNA. g The effect of RBFOX2 knockdown on SON-binding to PTBP2 pre-mRNA was measured by CLIP-qPCR assays with U87MG cells expressing control shRNA and RBFOX2 shRNA (n = 3). h The effect of SON knockdown on RBFOX2-binding to PTBP2 pre-mRNA was measured by CLIP-qPCR assays with U87MG cells transfected with control siRNA and SON siRNA (n = 3). The experiments were performed three times i PTBP2 minigenes containing the genomic DNA sequence from PTBP2 exon 9 to exon 11 downstream of the CMV promoter. Minigenes with wild-type (WT) or mutated (Mut) RBFOX2 binding motifs (yellow boxes) were cloned. Red asterisks indicate the mutated nucleotides. j RT-PCR analyses of exon 10 inclusion/skipping using WT and RBFOX2-binding site-mutated minigenes in control or SON siRNA-transfected U87MG cells. Data are representative of n = 3 independent experiments. Error bars in all graphs represent the standard deviation (SD) of tests. NS not significant, *p < 0.05, **p < 0.01. Statistical significance was determined by an unpaired two-tailed t-test. The number under each band in the Western blots indicates relative band intensity. Source data are provided in the Source data file.