Retinitis Pigmentosa Alleles, in PRPF31 and PRPF6, Effects on Pre‐Messenger RNA Splicing

Abstract

Retinitis pigmentosa (RP) is a genetically heterogeneous group of retinal disorders that affect 1 in 4,000 individuals in the United States. Patients with RP suffer from progressive degeneration of the light‐sensitive photoreceptors in the retina, which eventually leads to severe vision loss. The autosomal dominant form of RP (adRP) has been linked to eight splicing‐related proteins that affect the accuracy and efficiency of pre‐mRNA splicing. Among these splicing proteins, PRPF31 and PRPF6 are essential to the formation of the pre‐spliceosome. The purpose of this study is to examine the mechanisms by which adRP mutations in PRPF31 and PRPF6 affect pre‐mRNA splicing and to distinguish if these effects are widespread or locally more severe to the retina. This was done by constructing adRP allele orthologs, found first in human adRP patients, into two model organisms, Saccharomyces cerevisiae, and Mus musculus cell lines. First, we utilized S.cerevisiae to examine these mutations' effects on cell growth indicating a possible splicing defect and then examined splicing through in vitro splicing assays. Second, a mouse retinal ganglion precursor‐like cell line was utilized to identify various mRNAs effects of adRP mutants on splicing function and its effects on the phenotypic expression for PRPF6. In order to understand the role of pre‐mRNA splicing in photoreceptor gene expression and function, we have identified photoreceptor genes of interest whose pre‐mRNA splicing can potentially be affected by mutations in PRPF6. This is done by analyzing these genes of interest through splicing assays and immunofluorescent assays. Overall, this project adds a unique approach to studying how adRP alleles affect pre‐messenger RNA splicing.