Excision of introns from precursor messenger RNA by the spliceosome is a critical step in almost all human gene expression. This process is highly regulated, integrally linked with the transcription of genes and other processing events, such as polyadenylation and nucleotide modification. A better understanding of pre-mRNA splicing will be essential to further understand mechanisms that regulate splicing, that control patterns of alternative splicing, and that contribute to development, oncogenesis and retroviral infections. The mechanism by which the spliceosome recognizes the exact sites for the chemical events and how the reactions are catalyzed are not well understood. The long-term goals of this project are to understand interactions and rearrangements between active site components and the RNA ligands that are substrates for the catalytic reactions. Ample evidence argues for multiple rearrangements of factors and multiple recognition events at the branch site. Investigation of these events - which are not understood mechanistically - will elucidate interactions and rearrangements among active site components and may serve as a paradigm for other rearrangements and multiple recognition events that occur elsewhere in the spliceosome. This proposal focuses first on identification of all the proteins interacting with one site throughout the entire spliceosome cycle, with emphasis on a new approach that will allow indentification of molecules specifically contacting the RNA substrate only after splicing catalysis has occurred. Thus, this approach will allow a new view into the core of the spliceosome and the previously unexplored areas of transitions between the conformations for catalytic steps I and II that has not previously been possible. In addition, this allows investigation of interactions within unquestionably active spliceosomes. Further experiments will focus on interactions between the identified components and the RNA substrate, and on interactions of the identified components with other constituents of the spliceosome - with a particular bent as to mechanism by which these components interact to help juxtapose the reactants for the first chemical step. Finally, we will also investigate the recognition of non-conanical branch sites, which occur notably in some viral and a few cellular messages.
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