The biogenesis of mature eukaryotic RNA molecules requires posttranscriptional processing reactions that produce stable and functional mRNAs, rRNAs, snRNAs and tRNAs. Each of these RNA processing reactions provide targets for the regulation of gene expression, and thereby help to guide the proliferation and development of cells. Research into these RNA processing pathways has revealed steps within each that are used to regulate the rates of mature RNA production, yet little is known about how these RNA processing pathways are co-regulated to produce balanced levels of mature RNAs in response to changes in the cell's intracellular and extracellular environment. This proposal focuses on the roles of nuclear proteins implicated in the regulation of poly (A)+ mRNA and rRNA levels in S. cerevisiae. The yeast protein Rrp6p is a nuclear riboexonuclease homologous to an autoantigen produced in patients suffering from Polymyositis Scleroderma Overlap Syndrome (PM-Sc1), as well as to the Werner's and Bloom's syndrome proteins implicated in premature aging. Mutations in Rrp6p cause defects in rRNA processing and ribosome biogenesis. Rrp6p also plays a role in a nuclear mRNA degradation pathway, since loss of its function stabilizes an intermediate in the mRNA polyadenylation pathway. The experiments proposed here seek to determine the biochemical mechanism and physiological function of Rrp6p. The relationship of Rrp6p to other proteins will be determined using affinity purification and genetic techniques. Other proteins, whose connection to Rrp6p stems from our studies, include core components of an RNA processing complex called exosome, a subunit of RNA polymerase III and a riboexonuclease implicated in rRNA processing and mRNA nucleocytoplasmic transport. Rrp6p will be studied as a pure protein and complexed with its interacting partners. These studies will be carried out to determine which domains of Rrp6p are required for its ability to bind and hydrolyze RNAs, as well as which domains are required to interact with other proteins. The substrate specificity of Rrp6p will also be analyzed to determine what features of its RNA substrates are required for recognition by the enzyme and what features of the enzyme and its substrates are necessary for its ability to distinguish between mRNAs and rRNAs. These studies will illuminate the enzymatic properties of Rrp6p, as well as its role in nuclear mRNA degradation and rRNA processing. Moreover, the results of these experiments should provide basic knowledge regarding the functions of the PM-Sc1 autoantigens and the Bloom's and Werner's syndrome proteins, thereby contributing to an understanding of the processes leading to autoimmune disease and aging.
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