The overall goal of the proposed research is to obtain detailed mechanistic understanding of the multi-component complex formed by TRAMP (Trf4/Air2/Mtr4 Polyadenylation) and the nuclear exosome. This TRAMP-exosome machinery plays key roles in the regulation of eukaryotic gene expression. TRAMP consists of three subunits that are highly conserved in eukaryotes, a non-canonical poly(A) polymerase, a Zn-knuckle protein, and an RNA helicase. The eukaryotic nuclear exosome consists of eleven units, which are also highly conserved in eukaryotes, and include two functional 3'to 5'nucleases, Rrp6p and Rrp44p. TRAMP marks RNAs with short poly(A) tails, and resolves RNA secondary structures, while the exosome degrades the RNAs marked by TRAMP. Despite pivotal roles in RNA metabolism, it is not understood how the components of the TRAMP-exosome machinery coordinate their functions during RNA processing, and how RNA substrates are identified. In our proposal, we address these problems using biochemical means. Using a reconstituted TRAMP-exosome system, we will delineate which exosome and TRAMP components and activities are critical for the exosome stimulation by TRAMP, and for effects of the exosome on TRAMP function. To understand RNA processing by the TRAMP-exosome machinery at the level of individual reaction steps, we will devise a mechanistic, quantitative framework for the reaction. We will then examine how the TRAMP-exosome machinery identifies RNA targets. To this end we will investigate how hypomodified tRNAiMet is distinguished from correctly modified tRNAiMet, how the TRAMP-exosome machinery unfolds the RNAs, and whether and how RNA length and secondary structure affect the TRAMP-exosome machinery. We anticipate our studies to provide a new level of mechanistic understanding of the TRAMP-exosome machinery, and novel, significant insight into the molecular basis of nuclear RNA metabolism.
Defects in RNA processing have been linked to many diseases including autoimmune defects, neurodegenerative diseases, and cancer. To examine the molecular basis for these diseases and to guide the development of potential therapeutic agents, we propose to delineate the molecular function for the TRAMP complex and its interaction with the nuclear exosome, which are pivotal for quality control and correct processing of many cellular RNAs.
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