PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects
Mutations in the carboxy terminal of the core spliceosome factor PRPF8 cause autosomal dominant retinitis pigmentosa (RP) 13. Comprehensive cellular, biochemical, and molecular investigations of iPSC-derived retinal organoids, retinal pigment epithelium (RPE) and kidney organoids from four patients carrying the pathogenic PRPF8 RP type 13 c.6926A>C (p.H2309P) heterozygous missense mutation, revealed retinal tissue-specific effects including lower splicing specificity, ciliary abnormalities, altered apical-basal polarity, rod degeneration and loss of photoreceptors. The p.H2309P mutation affected the 5' splice site recognition by PRPF8 of transcripts encoding ciliary proteins, altered spliceosome kinetics and organisation of nuclear speckles as well as PRPF8 binding to spliceosomal U6 snRNAs and snoRNAs, leading to accumulation of unspliced poly A+ mRNAs specifically in RPE cells and retinal organoids. Together these data provide the most comprehensive characterisation of splicing factor causing RP disease, providing molecular insights into the tissue specificity of pathomechanisms and informing future therapeutic approaches. Overall design: Control and patient-specific ROs were subjected to single-cell RNA-Seq at day 210 of differentiation.