Fig. 7

Enhancement of splice-site strength renders the CDC20 mini-gene resistant to PRPF8 depletion. a A mini-gene containing a full-length intron flanked by two exons within the CDC20 gene. This particular intron is retained following PRPF8 depletion as determined by RNA-sequencing experiments and the corresponding coverage plot is shown. Both the 5′ splice site and the 3′ splice site encompassing the exon–intron and intron–exon boundaries are weaker than the corresponding consensus sequences and the sequence of 5′ and 3′ splice mutant constructs are indicated with changes marked in red. RPKM Reads per kilobase of exon per million reads mapped, WT wild type. b, c Enhancement of splice site strength renders the CDC20 mini-gene resistant to PRPF8 depletion. PRPF8 depletion strongly suppresses removal of this intron in the CDC20 mini-gene (left panel). Alteration by site-directed mutagenesis of the weak 5′ splice site into a strong consensus sequence leads to efficient removal of this intron in PRPF8-depleted cells, in a manner indistinguishable from control siRNA-treated cells (middle panel). Strengthening of the polypyrimidine tract also allows efficient removal of this intron from the CDC20 mini-gene in PRPF8-deficient cells (middle panel). Plots in (c) represent quantification of band intensity of spliced and unspliced product using ImageJ (NIH) and represent the mean percentage of spliced mRNA from three independent experiments ± standard error of the mean. Statistically significant pairwise comparison is indicated (***p < 0.001); n.s. not significant