Project Summary/Abstract RNA Silencing is a blanket term for a diverse group of biological processes in which small RNAs, including PIWI-interacting RNAs (piRNAs), microRNAs (miRNAs), and small interfering RNAs (siRNAs) silence genes. This proposal aims to leverage my group’s expertise in structural biology and biochemistry to address outstanding questions that stand as roadblocks to advancing the current understanding of RNA Silencing biology. Specifically: 1) piRNAs, essential for defending the genome against transposable elements (TEs), have been studied extensively using molecular genetics, but how piRNA-associated factors work together to produce the potent TE-silencing observed in animals remains unclear. We will address this knowledge gap by discovering the structures and biochemical activities of RNA-protein complexes central to piRNA function. 2) miRNAs are ubiquitous regulators of cellular gene expression in mammals, but how miRNAs themselves are regulated has only recently come to light. We will advance this emerging area by determining the structural basis for targeted ubiquitination and destruction of miRNA- protein complexes by the recently discovered ZSWIM8 Cullin-RING E3 ubiquitin ligase. 3) Although the canonical function of miRNAs is to silence targeted mRNAs, certain RNA viruses have evolved mechanisms to repurpose cellular miRNAs to instead stabilize their viral genomes and stimulate viral production. The mechanisms by which miRNA complexes are redirected to achieve these non-canonical functions are poorly understood. We are addressing this knowledge gap by investigating interactions between Hepatitis C Virus (HCV) and the liver-specific miRNA miR-122, with a focus on understanding the structures of miR-122–HCV RNA complexes that modulate HCV translation to enable replication of HCV RNA. 4) One of the oldest and most intriguing mysteries of the RNA Silencing field is that, in some cases, silencing RNAs readily pass between animal cells. 25 years after this discovery, the mechanisms for RNA transport into animal cells remain unclear. We are addressing this shortcoming by determining the structural basis for RNA recognition and transport by SID-1, a broadly conserved integral membrane protein that transports double-stranded RNA into animal cells. The combined studies are expected to produce insights necessary to overcome the major knowledge barriers described above, with implications for understanding human health, fertility, viral infection, and the advancement of RNA-based therapies.