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

PRPF 8蛋白是一种高度保守和普遍表达的前mRNA剪接因子，是U 5小核糖核蛋白颗粒（U 5 snRNP）的组分，其在剪接体的催化中心起作用。人类PRPF 8的突变导致常染色体显性视网膜疾病，称为视网膜色素变性（RP）。RP最突出的病理是感光细胞的损失和视网膜色素上皮（RPE）的退行性变化，其最终导致失明。我们最近已经表明，PRPF 8中的大多数RP突变损害了与U4/U6双snRNP特异性蛋白SNRP 200的结合，从而消除了U4/U6/U 5三snRNP的形成。但令人惊讶的是，Y2334 N突变仅引起选定转录物的错误剪接，而不影响三-snRNP的形成。这一观察结果的一个可能原因是PRPF 8与特异性前体mRNA的相互作用可能被Y2334 N突变削弱。或者，不同的序列和/或结构特征可能存在于视网膜特异性转录本中，这使得它们更容易受到有限数量的功能性PRPF 8的影响。视网膜特异性剪接变化在疾病的发病机制中起重要作用，并且受影响的途径代表潜在的治疗靶点，这是非常合理的。为了测试这一点，我们将首先开发一种适合人类剪接因子RP的疾病模型。为此，我们建立了RPE细胞的培养和CRISPR/Cas9编辑，以修饰和表达GFP标记的PRPF 8，其具有来自其内源基因座的RP突变Y2334 N。为了量化患病RPE细胞中WT PRPF 8及其Y2334 N突变体之间RNA-蛋白质相互作用和剪接效率的变化，我们将开发定量iCLIP程序和分析管道。同时，我们将对患病的RPE细胞进行RNA-Seq，以量化RNA丰度、剪接效率和可变剪接异构体的总体变化。这将揭示剪接抑制是否确实具有选择性，并确定受Y2334 N突变影响的外显子、RNA靶点和分子途径，这些可能有助于RP的发生和发展。iCLIP和RNA-Seq数据的整合以及对受影响的靶基因、剪接位点和内含子的计算检测可以揭示视网膜特异性剪接特征。这将提供新的机制的见解，核心剪接因子是如何能够调节选择的基因的选择性剪接，并帮助了解这些转录物的RP突变的敏感性升高。最后，将在患病RPE细胞中对具有疾病促进潜力的选定靶基因或同种型进行功能验证。

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.