Structure and Mechanism of Gene Regulation by Small Noncoding RNAs

In humans, microRNAs (miRNAs) of approximately 22 nucleotides (nt) are key components of mature RNA- induced silencing complexes (RISCs) and play essential roles in silencing specific genes. Our structural studies of human Argonaute proteins (AGOs) have revealed important differences among them, leading to several discoveries, including the identification of tiny guide RNAs (tyRNAs). TyRNAs are defined as guide RNAs of 17 nt or shorter that are associated with AGO proteins. We also uncovered a tyRNA biogenesis pathway driven by specific exonucleases, which generate 14-nt tyRNAs from AGO-associated miRNAs that are 21–23 nt in length. Among these tyRNAs, some, referred to as "cleavage-inducing tyRNAs (cityRNAs)," can catalytically activate AGO3. Despite these breakthroughs, the study of tyRNAs is still in its infancy, with significantly fewer publications compared to research on miRNAs. Meanwhile, more evidence has emerged that the four human AGOs play unique roles beyond conventional gene silencing. This project aims to address these gaps by focusing on five key topics: 1. Both AGO2 and AGO3, when loaded with cityRNA, appear to directly recognize the sequence upstream of the tyRNA target site (UTy), thereby influencing their target mRNAs differently from miRNAs. Our previous study demonstrated that cityRNA-mediated gene silencing relies heavily on target cleavage rather than translational repression or mRNA destabilization. However, the molecular mechanism of target recognition by cityRISCs remains unknown. We aim to address this knowledge gap through structural and functional studies. 2. Poly(A)-specific ribonuclease (PARN) processes miRNAs to approximately 22 nt in length and selectively degrades specific mRNAs, but the molecular basis of these activities is poorly understood. We aim to elucidate these mechanisms by determining the structure of the PARN-AGO complex bound to a miniature mRNA. 3. During duplex loading, AGOs recognize the less thermodynamically stable end of the duplex to capture the 5′ end of one strand as the guide, forming the mature RISC. The molecular mechanism for this asymmetric guide strand selection remains unclear. We will determine the cryo-EM structures of each step in the RISC assembly process. 4. Single-point mutations in AGO1 and AGO2 have been linked to neurodevelopmental disorders (NDDs). Our data show that specific 3′→5′ exonucleases trim miRNAs associated with these NDD-related AGOs into unusually short tyRNAs. To understand the impact of these mutations, we will determine the cryo-EM or crystal structures of NDD-relevant AGO mutants loaded with miRNAs and atypical tyRNAs. 5. Our structural studies have revealed distinct differences among the four human AGOs. To further characterize these differences, we will determine the structures of the four AGOs using the same set of guide and target RNAs. These proposed studies will explore a broad range of cellular events regulated by conventional and novel small noncoding RNAs. The outcomes will provide valuable insights and establish a solid foundation for the development of RNAi-based therapeutics. Project Number: 1R35GM161485-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Kotaro Nakanishi | Institution: OHIO STATE UNIVERSITY, Columbus, OH | Award Amount: $419,674 | Activity Code: R35 | Study Section: Maximizing Investigators' Research Award - E Study Section[MRAE] View on NIH RePORTER: https://reporter.nih.gov/project-details/11259628