Fig. 7: PRPF8 iCLIP provides information on the interaction between PRPF8 and pre-mRNA.

A PRPF8 does not selectively bind to 3’ splice sites. The 3’SS strengths of bound pre-mRNA fragments are normally distributed, as would be expected for random sequences. PRPF8 selectively binds to 5’SSs. The biphasic distribution of 5’SS strengths has two maxima (approximate maxent5 scores of 2 and 9) separated by a local minima. These were categorised into three 5’SS strengths: weak (maxent5 score <3), strong (maxent5 score > 8), and intermediate (maxent5 score between 3 and 8); (B) Histograms showing fold changes (RP13/RP13-Cas9) of weak and strong 5’SSs. KiOs and iPSCs histograms have more pronounced peaks with low fold change values. RPE and ROs histograms have flatter distributions with a greater proportion of the values present in the tails of the curve. The kurtosis of the ROs and RPE graphs is lower than iPSCs and KiOs (Fig. S10C); (C) Gene set enrichment analysis of differentially bound weak and strong 5’SSs in ROs showing enrichment for transcripts encoding proteins mediating transcription and ciliary functions (e.g., RNA polymerase II transcription, cilium assembly, cargo trafficking to periciliary membrane); (D) Gene set enrichment analysis of differentially bound weak and strong 5’SSs in RPE. Direction down and up refers to positive and negative fold changes in (RP13/RP13-Cas9), respectively. GSEAPY interface for Enrichr with Fisher’s exact test was used in (C, D); (E) Gene sequence plot showing differential inclusion of nucleotides at weak 5’SSs with upregulated or downregulated binding to PRPF8 in ROs (B).