Abstract 1721 Determining the Effects of Pre-messenger RNA Splicing in Retinitis Pigmentosa

Retinitis Pigmentosa (RP) is a retinal disorder that affects as many as 1.5 million people worldwide. Patients with RP experience gradual vision impairment leading to possibly complete blindness due to the degradation of photoreceptor cells. The autosomal dominant form of Retinitis Pigmentosa (adRP) is associated with eight pre-messenger RNA splicing factor proteins that help in the assembly of the spliceosome, including PRPF31 and PRPF6. The purpose of this study is to characterize and examine the mechanisms by which adRP mutations in PRPF31 and PRPF6 affect pre-mRNA splicing in two model organisms, Saccharomyces cerevisiae and Mus musculus cell culture. We constructed highly conserved adRP allele orthologs, found in human adRP patients, and then introduced them into our model systems. First, we utilized S.cerevisiae to monitor the mutations' effects on cell growth through the use of growth assays. We discovered that our mutations of interest generally did not cause a phenotypic effect. As a result, we shifted focus onto a mouse retinal ganglion precursor-like cell line, 661W, to study the effects of the mutations on cell morphology, protein delocalization and to identify if these mutations display an effect on pre-mRNA splicing. This was done by monitoring growth rates and analyzing the RP alleles through splicing assays, western blots, and immunofluorescent assays. We will present our current findings on how adRP alleles affect cell morphology and pre-messenger RNA splicing. This work was supported by The Welch Foundation, Max and Minnie VoelckerFoundation, and the WheelerFoundation.