Our long-term interest is in the mechanisms that control the specificity and fidelity of mRNA splicing and export. We study these problems in the budding yeast Saccharomyces cerevisiae in order to exploit its enormous genetic potential. Moreover, our work over the last 20 years has been instrumental in validating the principle - for mRNA splicing - that the basic steps in gene expression are remarkably conserved from yeast to mammals. In the last funding period, we have focused in particular on the role of ATP-driven rearrangements promoted by the DEAD-box family of proteins. A unifying hypothesis that has emerged from our studies is that mRNPs are remodelled extensively via the exchange of RNA:RNA or RNA:protein pairing partners; in some cases, these pairing partners are mutually exclusive. Future experiments will test the notion that these NTP-dependent exchanges are not only physically and temporally correlated, but are functionally interdependent, i.e. coupled. In addition to explicitly addressing the dynamic design principle of the spliceosome's catalytic core, these studies will suggest testable mechanisms for the integration of the major steps of gene expression with one another. Finally, we will exploit our recent development of highthroughput microarray technology to assess the genome-wide impact of specific mutations and environmental stresses on the full set of 250 intron-containing genes. Despite the widely-held notion that splicing in budding yeast is not significantly regulated, our initial analyses suggest that splicing in this single-cell organism is in fact subject to complex combinatorial control. Using a blend of biochemical, genetic, and cell biological approaches, we propose three Specific Aims: 1. To Test a Comprehensive Model for Catalysis and Fidelity at the Second Step of Splicing 2. To Identify Coupled Steps in the Sequential Remodelling of mRNPs that Confer Export Competence 3. To Identify Regulated Pathways in RNA Processing Using Genome-wide Analysis ? ?
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