RNA decay is a key mechanism of gene regulation in human cells, controlling RNA abundance and responses to stress. The goal of this proposal is to determine the molecular basis for stress- activated RNA decay mediated by the ubiquitous human protein kinase/endoribonuclease RNase L (Aim 1 and Aim 2) and to build tools to overcome the considerable experimental limitations in studies of this pathway (Aim 3). RNase L activates the production of interferons and cytokines, and inhibits a broad spectrum of human viruses, so a large body of research focuses on its antiviral effects. RNase L is a tumor suppressor gene in prostate cancer and is an essential mediator of adipocyte differentiation and apoptosis, making it an attractive target for diagnostics and treatment of a large number of human diseases. However, a part of the challenge with these applications, and a major challenge in understanding the RNase L-mediated RNA decay, is our limited knowledge of the molecular structures and mechanisms controlling this pathway. Our proposal aims to address these limitations by obtaining a detailed structural and molecular understanding of the key proteins and protein complexes responsible for RNA cleavage. This proposal is focused on structural and biochemical studies of RNase L and of two unconventional human RNA polymerases OAS1 and OAS3 required for activation of RNase L. During preliminary work described in our recent publications, we determined several crystal structures of these proteins or their domains. This progress and our established track record in the field of signal transduction and protein/RNA recognition position our laboratory ideally for structural and functional analysis of RNA decay mediated by RNase L. We propose three independent but complementary specific aims. In Aim 1 of this proposal we will elucidate the molecular mechanism by which OAS1 and OAS3 synthesize 2-5A, a specific cofactor of RNase L required for activation of RNA decay. This work is important because it will lay the foundation for rational studies of the machinery for synthesis of 2-5A and for therapeutic targeting of this machinery in diseases. In Aim 2 we will determine the crystal structure of RNase L. This work will provide a detailed molecular understanding of the unique protein kinase/RNase driving stress-activated RNA decay in human cells. In Aim 3, we will engineer light-emitting reporters of 2-5A, which will enable rapid and specific detection of 2-5A synthesis in vitro and in vivo. This work will eliminat a significant obstacle in the field due to the lack of tools to monitor the activation of the RNaseL pathway specifically and non-invasively. The long-term goal of our work will be to understand the mechanism of RNase L-mediated RNA decay by understanding structures, functions, and regulation of the molecular components in this pathway.