Regulation of alternative splicing by PTB and cofactors. (360G-Wellcome-077877_Z_05_Z)

We are investigating the mechanisms of alternative pre-mRNA splicing in three model systems in which the hnRNP protein polypyrimidine tract binding protein (PTB), plays a central role as a repressor. This programme will focus upon PTB, in particular analyzing its unusual role in effecting smooth muscle specific splicing of the a-tropomyosin gene, which contrasts with its more common role as a widely active repressor. · The first part of the proposal involves analysis of the mechanism of PTB-mediated repression of model pre-mRNAs. We aim to analyse the mechanistic basis of PTB-mediated splicing repression, identify additional regulators that are responsible for SM-specific regulation, analyze interactions between PTB and the cofactor raver1 that affect alternative splicing, and dissect the function of repressor domains in both proteins. · The second part of the proposal involves the application of quantitative proteomic approaches, as part of the current attempts to understand alternative splicing on a global basis. One approach will use proteomic approaches to characterize alternative splicing events that are affected by perturbations in the levels of PTB or other splicing regulators. The second application will be to characterize "cellular splicing codes" comprised of the relative levels of nucleoplasmic RNA binding proteins.