PRPF8-RNA interactions in Retinitis pigmentosa - novel disease models and quantitative iCLIP to study cause and consequence of splicing errors in neurodegenerative disease

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The PRPF8 protein is a highly conserved and ubiquitously expressed pre-mRNA splicing factor and a component of the U5 small ribonucleoprotein particle (U5 snRNP), which functions at the catalytic center of the spliceosome. Mutations in human PRPF8 cause the autosomal dominant retinal disorder called Retinitis pigmentosa (RP). The most prominent pathology of RP is a loss of photoreceptor cells and degenerative changes in the retinal pigment epithelium (RPE), which eventually lead to blindness. We have shown recently that most RP mutations in PRPF8 impair binding to the U4/U6 di-snRNP-specific protein SNRP200 and thus abolish formation of the U4/U6/U5 tri-snRNP. But surprisingly, the Y2334N mutation caused mis-splicing of only selected transcripts without affecting tri-snRNP formation. One possible reason for this observation is that the interaction of PRPF8 with specific pre-mRNAs might be impaired by the Y2334N mutation. Alternatively, distinct sequence- and/or structural features might exist in retina-specific transcripts, which render them more susceptible to limiting amounts of functional PRPF8. It is highly plausible that retina-specific splicing changes play an important role in the pathogenesis of the disease and that the affected pathways represent potential therapeutic targets. To test this, we will first develop a suitable disease model for human splicing factor RP. For this we established the culture of RPE cells and CRISPR/Cas9 editing to modify and express GFP-tagged PRPF8 with the RP mutation Y2334N from its endogenous locus. To quantify changes in RNA-protein interactions and splicing efficiencies between WT PRPF8 and its Y2334N mutant in diseased RPE cells, we will develop a quantitative iCLIP procedure and analysis pipeline. In parallel, we will perform RNA-Seq of diseased RPE cells to quantify global changes in RNA abundance, splicing efficiencies and alternative spliced isoforms. This will reveal whether splicing inhibition is indeed selective and identify exons, RNA targets and molecular pathways that are affected by the Y2334N mutation and which may contribute to the onset and development of RP. Integration of iCLIP and RNA-Seq data and computational inspection of affected target genes, splice sites and introns may reveal retina-specific splicing characteristics. This will provide novel mechanistic insights into how a core splicing factor is able to regulate alternative splicing of selected genes and help to understand the elevated sensitivity of these transcripts to RP mutations. Finally, selected target genes or isoforms with disease-promoting potential will be functionally validated in diseased RPE cells.