This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).
Intellectual Merit: DExD/H-box proteins are found in every living organism. They generally use the energy from hydrolyzing ATP to unwind double-stranded RNA or DNA to single strands in vitro. They also share several properties including a common structure of their helicase core domains. However, for the vast majority of RNA-dependent DExD/H-box proteins, their mechanisms of action and physiological RNA target substrates are unknown. This project focuses on Prp5, one of 8 DExD/H-box proteins required for pre-mRNA splicing within the spliceosome in the yeast Saccharomyces cerevisiae. Prp5's ATP-dependent activities are important for monitoring the accuracy of splicing and essential for forming the prespliceosome, an intermediate in the assembly pathway leading to the active spliceosome that catalyzes splicing. In this project, a combination of genetic and biochemical assays will be used to test three hypotheses regarding Prp5's interactions with RNA. These three hypotheses address two critical issues fundamental to all RNA-dependent DExD/H-box proteins; how RNA binding regulates ATPase activity and identifying the target of an RNA helicase. The first hypothesis is that a region immediately adjacent to the helicase core regulates Prp5's ATPase activity by linking RNA binding to ATP hydrolysis. In vitro ATPase and RNA binding assays will be used to characterize kinetic parameters of RNA stimulation in proteins with mutations in this region. The second hypothesis is that RNA stimulation of Prp5 modulates splicing accuracy. Mutant proteins defective in RNA stimulation of ATPase activity will be assessed for their effects on splicing accuracy in vivo. The third hypothesis is that Prp5's helicase core binds U2 RNA (another spliceosomal factor), pre-mRNA or both within the spliceosome. Directed hydroxyl radical mapping and in vitro splicing assays will be used together to identify the RNA that binds the core and is therefore Prp5's target substrate.
Broader impact: This research project will have several outcomes important to advancing science as well as education and training. The results will increase our understanding of many DExD/H-box proteins and the biological processes in which they participate. Another project outcome will be development of a new research tool (directed hydroxyl radical mapping) to further the study of these proteins. Three training, educational and outreach activities will involve students from middle school to graduate level. Graduate and undergraduate students will conduct parts of the project. High school students will visit the lab and shadow lab members to observe research in action, and to discuss training and educational opportunities. Finally, a STEM (Science, Technology, Engineering and Mathematics) team comprised of university faculty and students as well as middle school teachers and science professionals will be formed to introduce new learning sessions to middle school students. These sessions will develop scientific concepts and critical thinking skills as well as illustrate career choices and provide role models. The long-term outcome of the educational, training and outreach activities is to increase the number and diversity of individuals pursuing STEM careers.
