RNA Ligases and RNA Repair

Sloan-Kettering Inst Can Research, New York NY

Investigators

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Abstract

PROJECT SUMMARY: RNA repair pathways rely on RNA ligases to maintain or manipulate RNA structure in response to purposeful breakage events inflicted during physiological RNA processing (e.g. tRNA splicing; mRNA editing) and under conditions of cellular stress (e.g., virus infection, unfolded protein response). The goal of this project is to illuminate the mechanisms and structures of enzymes that repair broken RNA ends. Our proposed studies embrace two RNA repair systems that exploit ATP-dependent RNA ligases. (1) Fungal and plant tRNA ligases are essential for tRNA splicing and for mRNA splicing during in the unfolded protein response. A multifunctional tRNA ligase enzyme heals and seals the ends of the tRNA exons via the sequential action of 2',3'-cyclic phosphodiesterase, 5' polynucleotide kinase, and ATP-dependent RNA ligase catalytic domains. The defining feature of fungal/plant tRNA ligases is their requirement for a 2'-PO4 to synthesize a 3'-5' phosphodiester bond. The structural basis for this requirement is uncharted. Because mammals rely on a completely different biochemical pathway of tRNA exon splicing, and mammalian proteomes have no homologs of the fungal ligase domain, we regard fungal tRNA ligases as promising targets for antifungal drug discovery. To advance that end, we propose to structurally characterize the catalytic domains of tRNA ligases from several fungal species, including the human pathogens Aspergillus fumigatus, Candida albicans, and Coccidioides immitis. (2) Naegleria gruberi RNA ligase (NgrRnl), is the founder of a new family of RNA nick-sealing enzymes ? found in bacteria, viruses, fungi, and protozoa ? in which a nucleotidyltransferase module (common to all ATP- dependent ligases) is fused to a signature N-terminal module not found in any other RNA ligase clade. Our structures of the NgrRnl·ATP·(Mn2+)2 Michaelis complex and the NgrRnl-(lysyl-N)?AMP·Mn2+ covalent intermediate suggest a two-metal mechanism of ligase adenylylation. We will use these structures to guide functional studies of the ligase reaction mechanism. To understand the basis for nick recognition and phosphodiester synthesis, we propose to capture structures of NgrRnl in complexes with nicked duplex substrates.