Functional dissection of core spliceosomal mutations causing Retinitis Pigmentosa.

Over 95% of human genes undergo pre-mRNA splicing, and alternative splicing of mRNA precursors represents a prevalent mode of gene regulation. Errors in this process are often the origin of disorders. Mutations affecting directly splicing factors, including core spliceosomal components, have been linked to various pathologies. Particularly intriguing are variants of the key spliceosomal subcomplex U4/5/6 tri-snRNP, associated with Retinitis Pigmentosa, one of the most common hereditary diseases affecting 1 in 3,000 individuals, leading to retinal degeneration and progressive blindness. Why these mutations lead to highly tissue-specific phenotypes, rather than general toxicity cause by a global block in splicing, remain unexplained.
The proposed research aims to increase our understanding of the molecular mechanisms underlying the effects of these mutations and shed light on the basis of the disease. To functionally dissect these variants, I combined spliceosomal network approaches (I) with genome-wide transcriptome analysis (II). Mechanistic insights derived from these analyses will helped to identify transcripts that are predominantly sensitive to these mutations and that could be behind their pathogenic effects. This work l allowed us to better understand the function of key splicing factorsand their contributions to Retinitis Pigmentosa.

The overall goal was to evaluate the mechanisms by which Retinitis Pigmentosa-related pathogenic variants of core splicing factors affect pre-mRNA splicing.
This project combined functional network approaches with transcriptome-wide RNA profiling and biochemical to systematically study mechanisms of regulation by natural variants of core splicing factors. Using pipelines for large-scale RNA profiling and network reconstruction, I will interrogate the functional effects of the in six U4/5/6 tri-snRNP-specific proteins which have been linked to Retinitis Pigmentosa. As result I have identified and later validated a set splicing events in genes with relevance for retina function or associated with retinal degeneration. Furthermore, I demonstrated that those events are sensitive to the levels or/and mutations in core splicing factors frequently mutated in patens with RP.
Furthermore, I while analyzing the data set of 300 KD of core splicing factors available in the lab I observed broad cross regulation between splicing factors via alternative splicing and proved that core splicing factors have distinct regulatory networks and might impact alternative splicing events by affecting other splicing factor or RNA binding proteins.

Transcriptome-wide RNA profiling of knock downs of splicing factors linked to autosomal dominant RP allowed identification of novel splicing events that could potentially explain the pathogenic effects of mutations. Moreover, those events could become a target for newly available therapies based on splicing modulation by modified antisense oligonucleotides or small compounds. Consequently, the discovery of potential targets for human genetic diseases represents unique opportunity to improve available treatments.
The broad cross regulation between splicing factors, which represents additional finding observed during the project indicates that interpretation and prediction of the effects of genetic alterations of the splicing machinery, requires understanding the complexity of the splicing self-regulatory network. This sheds the light on the intricate regulatory circuits of spliceosomal components and improves our understanding of splicing regulation.

Periodic Reporting for period 1 - SpliceCore (Functional dissection of core spliceosomal mutations causing Retinitis Pigmentosa.)

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