PROJECT SUMMARY The long-term mission of my lab is to decipher the critical pathways that sense nucleic acids as a danger, and the post-transcriptional gene regulatory components that facilitate the subsequent cellular response - with the goal that such knowledge significantly contributes to our scientific understanding of innate immunity in human health and disease. Research on innate immunity has traditionally focused on understanding the initiating triggers that ultimately modulate the transcriptional expression of a central set of interferons and cytokines. Much less is known about the post-transcriptional gene regulatory layer, which acts to refine innate immune activation at the RNA level – shaping gene expression to allow for a robust but finite host response while simultaneously preventing aberrant or pathogen- associated gene expression. This remains a striking gap in our understanding given that many aspects of host-pathogen interactions have at its core the detection and suppression of foreign nucleic acids, particularly from viruses. RNA-binding proteins (RBPs) can pre-program the sensitivity of cells to immunogenic stimuli, as well as being essential factors in the anti-viral response. Towards these interests, and in line with our outlined goals of the first cycle of our R35 funding period, we made substantial progress in our investigations on: 1)The characterization of essential and immune-relevant RBPs including ELAVL1 and the YTHDF protein family, 2) the discovery of widespread viral-host RBP interactions through our development of VIR-CLASP and characterization of their impact on viral replication, and 3) the identification and characterization of a novel small molecule catalytic inhibitor of the cGAS-STING pathway, given that we also model dynamic post-transcriptional regulation by examining how this primary cytosolic DNA sensor initiates a generalized innate immune response to perceived foreign nucleic acids. Taken together, my laboratory has grown in expertise in RNA/DNA binding protein biochemistry and -omic scale biology aimed directly at characterizing the essential roles of host RBPs and components of the cGAS pathway in conferring innate immunity. For this research renewal, we aim to expand our understanding within our established research program by pursuing the following major biological questions: 1)How do non-canonical cellular RBPs regulate and maintain host gene expression, especially during viral infection? 2)What are the unique host RBP-viral genome interactions that impact replication and virulence of related viruses within different intracellular environments during early infection? 3) What are the novel components that regulate and facilitate a non-canonical cGAS-dependent pathway promoting innate immune transcriptional activation and programmed DNA damage.