Additional file 7: of Regulation of constitutive and alternative mRNA splicing across the human transcriptome by PRPF8 is determined by 5′ splice site strength

Enhancement of 3′ 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. The 3′ splice site encompassing the intron–exon boundary is weaker than the corresponding consensus sequence and the sequence of 3′ splice mutant constructs are indicated with changes marked in red. b, c Enhancement of 3′ 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). Strengthening of the polypyrimidine tract 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 ± standard error of the mean from three independent experiments. Statistically significant pairwise comparisons are indicated (*p < 0.05, **p < 0.01). n.s. not significant, WT wild type. (PDF 135 kb)