The RNA interference (RNAi) and related pathways globally impact genome expression, govern diverse patho/physiological processes, and promise novel small RNA-based therapeutics for human diseases. The RNA-induced silencing complex (RISC) is the catalytic engine of RNAi, wherein single-strand siRNA guides Argonaute 2 (Ago2) RNase to catalyze sequence-specific cleavage of complementary mRNA. Thus, we will use a multidisciplinary approach to answer the following open and fundamentally important questions: 1) what are the mechanisms of RISC loading and activation? 2) what are the structural bases of dynamic RISC assembly? In Aim 1, we will combine forward genetic screen and biochemical fractionation & reconstitution to identify new RNAi factors, define the molecular composition of the Drosophila RISC loading complex (RLC), and elucidate the detailed mechanism by which the RLC transfers duplex siRNA from Dicer2-R2D2 complex to Ago2 to form inactive pre-RISC. In Aim 2, we will apply a novel ChemiC-Grid electron microscopy (EM) technology that we developed to investigate structural underpinnings of the dynamic process of RISC loading and activation. This cutting-edge technology allows for selective enrichment of recombinant RLC and RISC assembled on the siRNA-coated ChemiC-Grids and cryoEM imaging of these complexes at specific functional state at near physiological concentrations. These two highly complementary Aims will provide an integrated biochemical/structural framework for understanding the fundamental mechanisms of eukaryotic RISC assembly. The knowledge is essential to understanding human diseases in which these pathways are disrupted, and for developing novel therapeutics that either target or utilize these small bioactive RNAs.