Project Summary/Abstract Nearly every cell in the human body contains a set of programmable gene-silencing proteins named Argonaute. The natural function of Argonaute proteins is to mediate gene-regulation by microRNAs (miRNAs), small RNAs that contribute to cellular homeostasis during diverse physiological process, such as stem cell maintenance, fertilization, and heart development. Human Argoanute-2 (Ago2) can also be harnessed for experimental and therapeutic purposes through the introduction of small interfering RNAs (siRNAs), which are bound by Ago2 and used to direct the silencing of targeted genes. The overarching goal of this research is to understand how small RNAs are generated and used by Argonaute to silence genes. The rational motivating this work is that improved understanding of silencing processes will empower efforts to harness these mechanisms in a therapeutic setting and inform treatment of disease states resulting from aberrant miRNA function. Additionally, because miRNAs contribute in many facets of human biology, improvements in understanding function will enable researchers studying diverse aspects of human health and disease. Major projects include: 1) determining the structure of human Dicer, the enzyme that catalyzes the final step of miRNA biogenesis, with the goal of understanding how Dicer mutations drive diverse and devastating forms of human cancer; 2) identifying determinants of miRNA targeting with the goal of improving prediction of miRNA targets; 3) investigate the structure of miRNA- induced silencing complexes with a focus on understating how liquid:liquid phase separation contributes to gene-regulation by miRNAs; 4) establish the structural basis for mRNA cleavage by siRNAs, with the goal of providing novel insights for rational design of therapeutic siRNAs. The combined studies are expected to provide fundamental knowledge necessary for deciphering and controlling Argonaute- mediated regulation of human gene expression.