The most common form of editing in mammalian RNA is deamination of adenosine to inosine by ADAR1 and ADAR2. ADAR1p150, an interferon inducible adenosine deaminase, is also known to antagonize RNA interference (RNAi). To date, the mechanism by which this is carried out is unclear. For example, it is unknown if the embryonic lethality of ADAR1 knockout mice is related to antagonism of RNAi. Thus, I propose to study how ADAR1 antagonizes the RNAi pathway and if it interacts directly with RNAi components. Mutants of ADAR1 have been generated in the host laboratory. I propose to examine the activity of each mutant against RNAi in HEK 293 cells. These mutants will allow us to determine if RNA binding, localization or editing by ADAR1 is required for ADAR1 antagonism of RNAi. The effects on each branch of the RNAi pathway, siRNA and miRNA, will be elucidated using advanced techniques including miRNA detecting microArrays, cloning, dual-luciferase, and miRNA/siRNA reporters. Further, classical approaches of protein-protein interaction, and a recently optimized Tap-tagging approach, will be used to reveal if ADAR1 interacts directly with components of the RNAi machinery. This proposal supports collaboration between Dr. Bret Heale and Dr. Mary O’Connell. Dr. Heale received his PhD in June 2006 (USA) for his studies in the RNAi field, specifically related to siRNA and miRNA recognition of target sites, and in the course of his PhD published two first author publications (one in siRNA targeting and one in miRNA targeting) and authored a computer program to predict siRNA efficacy. Dr. Mary O’Connell (UK) has spent nearly twenty years working in the field of RNA editing, specifically examining ADARs. Thus, this proposal embodies the goals of the FP7 “People” work program to bring talented scientists to Europe, further European science, and to strengthen international ties.
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