| The process of pre-mRNA splicing involving the ligation of exons and removal of introns, constitutes a critical aspect of gene regulation. Splicing of a pre-mRNA into mRNA is brought about by an intricate interplay of small nuclear ribonucleoproteins (snRNPs) and accessory protein factors. Additionally, the process of alternative splicing provides biological systems with the capacity to increase protein diversity and also fine tune the cellular milieu in response to external cues. The importance of splicing is highlighted by several diseases that arise out of mutations that affect either the RNA or protein component of the splicing machinery.;This project aimed to identify the role of a core spliceosomal factor, Prp31 in a retinal degeneration disorder, RP11. Results showed that mutant Prp31 acts in a dominant negative manner to reduce the splicing efficiency in an in vitro splicing assay. The mutant protein failed to show an interaction with the Snu66 protein which in turn interacts with crucial components of the splicing machinery. These observations provide a plausible molecular mechanism for pathogenicity of mutant Prp31 protein. We also developed a Drosophila model for RP11 which successfully recapitulated the two main features of the disorder -- bimodal inheritance and incomplete penetrance. Drosophila Prp31 is essential for development of the fly eye and human Prp31 rescues the defects caused by siRNA mediated reduction of the endogenous Prp31 in Drosophila melanogaster.;This work also examined regulators of Tau Exon 10 splicing. Mutations affecting Tau Exon 10 splicing have been implicated in FTDP17 wherein patients show an imbalance in the tau isoforms containing or lacking Exon10. Using an RNA-protein interaction screen, PSF was identified as a regulator of Tau Exon 10 splicing. PSF has previously been implicated as a regulator of alternative splicing for substrates such as CD44; however this is the first evidence of its involvement in the splicing of a neuronal substrate.;Mutations in components of splicing machinery have been linked to several disease conditions. However the exact molecular mechanism of disease pathogenesis still eludes us in numerous cases. A detailed investigation of pre-mRNA splicing and its regulation will enhance our understanding of the basic mechanism involved. This knowledge will facilitate the development of strategies that can be used to circumvent events arising due to mis-regulation of splicing and alternative splicing. |
