Elucidating roles and mechanisms of double-stranded RNA-mediated pathways

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SUMMARY/ABSTRACT The goal of the proposed research is to define and characterize the long double-stranded RNA (dsRNA) encoded and expressed in animals, including humans, and the proteins that bind, modify, and process this dsRNA. A key, health-related, focus is how this endogenous dsRNA is discriminated from the long viral dsRNA that is recognized as foreign to trigger an innate immune response. Recent studies show that in both vertebrates and invertebrates, the RNA editing enzymes called Adenosine deaminases that act on RNA, or ADARs, deaminate endogenous dsRNA so that it will not trigger an aberrant immune response. The mechanism by which ADARs, and the editing sites they create, preclude activation of an innate immune response is unclear, and the proposed research is designed to fill this gap in knowledge. Using genetic screens and molecular approaches, the model organism C. elegans will be used to discover RNAs and proteins that lead to an immune response in strains lacking ADARs; biochemical approaches will be used to provide in-depth mechanistic insights. While mammals use the interferon pathway to mount an antiviral response, invertebrates lack this pathway, and instead, use RNA interference (RNAi) in antiviral defense. The enzyme Dicer is key to the antiviral RNAi pathway and is essential for cleaving viral dsRNA during the invertebrate immune response. Our prior in vitro studies indicate Dicer's helicase domain recognizes the ends of viral dsRNA as “nonself”, or foreign, and the proposed studies are designed to test this in vivo. The helicase domain of invertebrate Dicers is a fascinating molecular motor, and biochemistry, transient kinetic analyses, and structural biology, will be used to understand how it coordinates dsRNA cleavage, and ultimately passes small RNA products to downstream factors that enable gene silencing by the RNAi pathway. Modulation of Dicer's activity by accessory factors that interact with the helicase domain will be investigated. Differences in activities of the helicase domain of human Dicer and invertebrate Dicers will be explored to understand how this enzyme evolved as the immune pathways of vertebrates and invertebrates diverged.