Mammalian Pre-mRNA Splicing Mechanisms

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The long-term goal of this project is to elucidate the detailed molecular mechanisms by which intervening sequences or introns are removed from nascent RNA transcripts through the process of pre-mRNA splicing, an essential step in eukaryotic gene expression. Human genes are on average >90% intron, and a significant percentage of genetic diseases arise from mutations that that alter splice site choice . It has been estimated that ca. half of of human genes are additionally subject to alternative splicing, which significantly increases the complexity of the proteome encoded by our surprisingly small genome. This alternative splicing is often tissue-specifically or developmentally regulated. The extent to which a transcript is spliced is also subject to control - for example, altering the ratio of spliced and unspliced viral RNAis critical to the replication of HIV. Thus a detailed working knowledge of the splicing process will be essential if we are to understand not only the basic mechanisms of eukaryotic gene expression, but also how they relate to the complex processes of development, oncogenesis, human genetic disorders and the progression of retroviral infection. Studies in this proposal will address three important questions: (A) What is the detailed three-dimensional architecture of the human spliceosome, the macromolecular protein:RNA machine that mediates intron excision?; (B) What is the detailed three-dimensional architecture of the exon junction complex (EJC), a key regulator of spliced mRNA metabolism?; and (C) By what mechanism does the EJC remain stably bound to spliced mRNA? Techniques utilized will include (i) structure determination of purified complexes by cryo- electron microscopy (EM); (ii)labeling of those structures with EM-visible probes to map the locations individual components; (iii)a new methodology for determining relative protein stoichiometries by quantitative mass spectrometry; (iv)protein-protein interaction studies; and (v) determination of the kinetic and thermodynamic properties with respect to RNA and ATP binding by the protein thought to serve as the EJC anchor in the presence or absence of its binding partners.