Fig. 3

PRPF31 expression in patient-specific cells and effects on pre-mRNA splicing. a Gel electrophoresis showing the presence of a long mutant transcript (LM) isoform for the exon 11 deletion in patient-specific cells. The short mutant (SM) isoform is present only upon inhibition of NMD with puromycin (indicated by + ); b The bar graph shows wild-type PRPF31 mRNA in patient cells relative to controls from a, b. Data are representative of at least three independent repeats, RO retinal organoids; c Wild-type PRPF31 is significantly reduced in patient RPE cells and less notably in retinal organoids. The LM form and reduced SART1 is observed only in the patient RPE cells; d The bar graph shows wild-type PRPF31 levels in patient cells relative to normal cells quantified from c, n = 3; e, f Patient RPE cells and retinal organoids exhibit a notable defect in the alternative splicing of E1A minigene reporter. Schematic representation of alternative splice variants of the E1A reporter (e) and denaturing PAGE and autoradiography using a phosphoimager (f), n = 3; g Northern blot analysis showing the level of snRNAs in various normal and patient cells. Total RNA was isolated from each sample and snRNA levels were analysed by denaturing PAGE followed by Northern blotting using probes against U1, U2, U4, U5, U6 and 5S rRNA (top). The levels of snRNAs were quantified and normalised to the amount of 5S rRNA (bottom), n = 2. All error bars represent SEM