PROJECT SUMMARY In humans, microRNAs (miRNAs) are key components of mature RNA-induced silencing complexes (mature RISCs) involved in silencing specific genes. Recent research has uncovered a new class of smaller RNAs called tinyRNAs (tyRNAs), which are shorter than miRNAs and associate with AGO proteins to form tyRNA- induced silencing complexes (tyRISCs). A significant discovery is the existence of a tyRNA biogenesis pathway driven by specific exonucleases, resulting in the production of 14-nt tyRNAs from AGO-associated 21~23 nt miRNAs. Some tyRNAs, known as "cleavage-inducing tyRNAs (cityRNAs)," can catalytically activate Argonaute3 (AGO3). Despite these breakthroughs, the study of tyRNAs is in its infancy, with only a limited number of papers compared to miRNAs. The primary goal is to comprehensively understand the roles of tyRNAs, with three specific aims: Aim 1: Investigate the mechanism of target recognition and cleavage by cityRISC. AGO3 loaded with 14-nt cityRNA cleaved a complementary target RNA at low efficiency. Cleavage was enhanced when the target was extended by 9 nt on its 5' side, suggesting AGO directly recognizes the sequence of the extended target site. This challenges the conventional idea that the guide is essential for target recognition. Structural studies and RNA sequencing will provide insights into this unique target recognition. Aim 2: Explore the structural basis for neurodevelopmental disease-relevant AGO1s (NDD-AGO1s). NDD- AGO1s with specific mutations exhibit abnormal trimming of associated miRNAs. Cryo-EM structures of these mutant AGO1s will reveal structural alterations compared to wild-type AGO1. Additionally, the interaction between AGO1 and poly(A)-specific nuclease (PARN) will be examined to understand the distinct trimming patterns. Aim 3: Determine the silencing ability of NDD-AGO2-RISCs loaded with tyRNA (NDD-AGO-tyRISCs). The study will investigate whether NDD-AGO2 mutants exhibit similar miRNA trimming as NDD-AGO1s and assess the silencing capability of NDD-AGO2-tyRISCs. Co-localization of NDD-AGO2-tyRISC with AGO-binding proteins will be analyzed. Through these aims, the research aims to unravel the mechanisms of target recognition by cityRNAs, understand tyRNA pathology, and characterize NDD-tyRISCs, which may lay the groundwork for potential therapeutic applications.