CAREER: Structural Studies of Ski2-like RNA Helicases

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Intellectual Merit. RNA surveillance and decay pathways are essential for maintaining proper cellular function and viability. Many of these RNA decay pathways are mediated by Ski2-like RNA helicases, which account for 10% of all RNA helicases. A complete mechanistic understanding of how these helicases function to recognize and ensure appropriate decay of RNA substrates is considerably limited due to a lack of detailed structural and biochemical data. The goal of this project is to elucidate the molecular details of this important class of enzymes through crystal structure determination, supported by biochemical and biological analyses. An important focus of this project is the Ski2-like Mtr4 protein, a highly conserved and essential eukaryotic protein that plays a central role in nuclear RNA processing and decay. Mtr4 is required for ribosomal RNA biogenesis, and also targets a wide array of RNAs for degradation as part of the TRAMP (Trf4-Air2-Mtr4 polyadenylation) complex. A second target is Ski2, the cytosolic counterpart of Mtr4, which mediates degradation of mRNA transcripts. The specific components of this project include: determination of high-resolution crystal structures; identification of important protein-RNA interactions; and characterization of a novel arch domain that is unique to Ski2-like helicases.Broader Impacts. The broader impacts of this project include extensive training of undergraduate and graduate students in structural biology. A new course in structural biology has been developed which provides rigorous hands-on training in protein structure determination. The departmental senior-level biochemistry laboratory course has been redesigned to accommodate a new and growing biochemistry major. In an effort to introduce research-based experiments into the laboratory course, student groups will be assigned individual protein expression and purification projects that will enter the pipeline for crystallization trials. High school students will participate in a summer research experience to introduce fundamental concepts of crystallization and structure determination. This broad educational approach will give widespread exposure and direct access to the rapidly expanding field of structural biology, which has historically been less accessible to geographically remote/smaller institutions. The successful implementation of a new program in structural biology at a smaller university campus, with the full capacity to perform high-end crystallographic research, provides a template that may be reproduced in similar environments.Co-funded by Genes and Genome Systems in the Division of Molecular and Cellular Biosciences and by the Experimental Program to Stimulate Competitive Research.