PROJECT SUMMARY In eukaryotic cells, gene expression is regulated at multiple levels, including post-transcriptional gene silencing, where microRNAs (miRNAs) bind to complementary target RNAs and cause translational repression. Argonaute (AGO) proteins and miRNAs form RNA-induced silencing complexes (RISCs), the core players in gene silencing. Humans have four AGO proteins, AGO1-4, which share a high sequence identity, and the majority of miRNAs bound are common across all AGOs. Therefore, it has been thought that the four AGOs work redundantly. Nevertheless, an increasing number of studies have found that each AGO has its unique roles in various biological processes and diseases in addition to gene silencing. Although the interaction of all four AGOs with miRNAs has been well characterized, little is known about how each RISC recognizes its target RNAs. Elucidation of this recognition will provide insight into the unique roles of each AGO. Meanwhile, characterization of RISC and target interactions will facilitate target prediction accuracy by improving prediction algorithms, which will take account of not only the complementarity between guide and target but also the type of AGO and target interaction. In this proposed study, we will pursue the following specific aims. In Aim 1, we will use cryo-electron microscopy and X-ray crystallography to determine the structures of all four homogenously purified RISCs with the same guide and target RNAs, which will provide insight into the differences in target recognition by the four AGOs. In Aim 2, to clarify these differences, we recently developed a novel SHAPE-based technique which allows us to visualize the conformational dynamics of target RNA bound to RISC. The method will enable us to characterize this interaction within the RISC binding channel and its periphery at a single-nucleotide resolution and can be expanded to understand how RISCs recognize guide- binding sites buried within highly structured target RNAs. In Aim 3, we will first use mass spectrometry to identify the unique protein binding partners of each AGO and their specific sites of interaction. Then, we will use tandem immunoprecipitation, followed by RNA sequencing, to determine how the binding of these proteins influences the target specificity of each AGO and directs their functionality towards alternative cellular events. The outcome from this study will provide a solid foundation for fields beyond gene silencing and enable the development of new strategies for higher accuracy guide-RNA drug design in therapeutic applications.