Structural, Functional and Genetic Analysis of Archaea RNA Ligase

Intellectual merit: Living cells are under constant attack by various environmental and cellular agents that can induce breakage of DNA and RNA. If not repaired, this damage can lead to changes in gene expression, mutation, or even cell death. The molecular mechanisms of cellular repair of damaged RNA molecules are poorly characterized, especially in comparison to all that is known regarding repair of damaged DNA. One of the few established facts concerns the central role of RNA ligase in RNA repair. This enzyme recognizes breaks in an RNA chain and joins the two ends together. Although RNA ligases have been identified in many organisms, including humans, their mechanisms remain poorly understood. The recent discovery of a novel RNA ligase (Rnl3) in Archaea, unicellular organisms that have remarkable biosynthetic capacities and thrive under extreme environmental conditions, opens an exciting new avenue for exploring the role of RNA ligase in cellular RNA repair and processing. Archaeal enzymes are generally much more stable than enzymes derived from other organisms, and are therefore ideal for biochemical and structural studies. A central goal of this project is to identify the physiological targets of Rnl3, and thereby the biological processes affected by the cellular RNA ligation pathway. Genetic approaches will be used to identify these targets in the organism, and ligation assays will be carried out in vitro to further characterize specific RNA substrates. Structural and functional analyses will be performed to elucidate how Rnl3 recognizes breaks in target RNA(s) and catalyzes the joining of the two ends of the damaged RNA. The fundamental knowledge the project will generate concerning the function of archaeal RNA ligase is expected to be relevant to the regulation, repair, and recombination of cellular RNA in other organisms, including humans. Broader impact: The project will provide fundamental new insights into the structure, function, and evolution of RNA ligases, and will thereby advance understanding of how cells respond to, recognize, and repair RNA damage. Furthermore, the project's focus on thermostable RNA ligases is expected to benefit biotechnology, as RNA ligases are widely used in molecular biology applications. The multi-disciplinary nature of the project will provide integrative training in cutting-edge techniques in RNA and protein biochemistry, structural biology and molecular genetics to graduate and undergraduate students who participate in the research work. Undergraduate students, including women and members of under-represented demographic groups, will be recruited to participate in the project through the CURCA (Center of Undergraduate Research and Creative Activities) and CLIMB (Collaborative Learning and Integrated Mentoring in the Biosciences) programs sponsored by the University of Buffalo, the American Society of Pharmacology and Experimental Therapeutics and FASEB Minority Access to Research Careers. Each undergraduate will plan and carry out molecular cloning and biochemical assays to answer a scientific question and will present their research results at the University-wide research fair. Finally the PI will participate in outreach programs organized by the University of Buffalo Center for Educational Passport STEM (science, technology, engineering, and mathematics) designed to stimulate student interest in professional careers in the life sciences.