Fig. 4

HnRNP A1 promotes the skipping of exon 6 and exclusion of CCDC50 pre-mRNA. a CCDC50 splicing regulator was screened from a series of splicing factors using RT-PCR in HEK293 cells. GAPDH was used as the internal control. b Negative correlation between the expression level of CCDC50 exon 6 and HnRNP A1 was observed in ccRCC samples (r = − 0.361, p < 0.0001). c Minigene including the full sequence of exon 5, exon 6, exon 7 and flanking part of intron 5 and intron 6 was designed and constructed. d Regulation role of HnRNP A1 on CCDC50 splicing was validated by RT-PCR in (a) 786-O and (b) OS-RC-2 cells, after the minigene and HnRNP A1 or sh-HnRNP A1 plasmid were transfected. GAPDH was used as the internal control. e Regulation role of HnRNP A1 on CCDC50 splicing was comfirmed by western blot in (a) 786-O and (b) OS-RC-2 cells, after the HnRNP A1 or sh-HnRNP A1 plasmid were transfected. β-actin was used as the internal control. f Binding of HnRNP A1 with CCDC50-S and pre-mRNA sequences was confirmed by RNA immunoprecipitation (RIP) in 786-O cells