Fig. 2

hnRNP A1 binds downstream of the 5′ splice site of repressed exons. a Validation of hnRNP A1 knockdown by western blotting. b RT-PCR validation of hnRNP A1-regulated splicing events identified by RNA sequencing of hnRNP A1-depleted HeLa cells. Representative of two replicates. PSI percent spliced in; semi-quantification of bands on agarose gels by ImageJ. c Types of alternative splicing events regulated by hnRNP A1 identified by hnRNP A1 knockdown and RNA sequencing. Most splicing events regulated by hnRNP A1 are splicing of cassette exons. Most of the regulated cassette exons are repressed by hnRNP A1. d Distribution of hnRNP A1 iCLIP tags in cassette exons activated (green) or repressed (orange) by hnRNP A1 and the upstream and downstream exons. The analysis was based on 18 activated exons and 110 repressed exons found by RNA sequencing of hnRNP A1-depleted HeLa cells (n = 3). The hnRNP A1 crosslinking site density is higher across cassette exons repressed by hnRNP A1 than cassette exons activated by hnRNP A1, suggesting that hnRNP A1 exon repression is a direct mechanism, while hnRNP A1 exon activation is an indirect mechanism. There is no difference between the distribution of hnRNP A1 iCLIP tags in upstream and downstream exons of hnRNP A1-activated and hnRNP A1-repressed exons. The region immediately downstream of the 5′ splice site seems to be important for hnRNP A1-mediated exon repression. The level of hnRNP A1 crosslinking sites in exons not affected by hnRNP A1 knockdown is shown in purple. Shaded region reflects the 95 % confidence interval. e Maxent score for 3′ splice sites and the 5′ splice sites of hnRNP A1-activated, -repressed, or neutral cassette exons. *p value < 0.05. Wilcoxon rank sum test, p = 0.02278. ns non-significant. Error bars are standard error of mean