# Structural and molecular basis for cityRNA (cleavage-inducing tiny RNA)-directed RNA cleavage by AGO3 > **NIH NIH R01** · OHIO STATE UNIVERSITY · 2022 · $199,982 ## Abstract Summary/Abstract of Parent Award Project MicroRNAs (miRNAs) are 19~23 nucleotide(nt)-length noncoding RNAs that are loaded into Argonaute proteins (AGOs) to assemble the RNA-induced silencing complexes (RISCs). Among four human AGOs, only AGO2 has been shown to have slicer activity when target RNAs are fully complementary to the guide. We recently discovered that AGO3 becomes a competitive slicer of AGO2 when loaded with 14-nt tiny RNAs (tyRNAs) of miR-20a whose 3’ 8~9 nt are deleted. In contrast, AGO2 drastically decreased the slicing activity when loaded with those tyRNAs. Our findings demonstrate that AGO2 and AGO3 have different optimum lengths of guide RNA for slicing activity. We defined such tyRNAs as cleavage-inducing tyRNAs (cityRNAs). We recently identified several 3’→5’ exonucleases in vitro and identified interferon-stimulated exonuclease gene 20 (ISG20) that can trim AGO3-bound 23-nt miR-20a to 14 nt and thereby activate the RISC for RNA cleavage. Although previous studies reported that viral infection increases the ISG20 level, little is known about the role of ISG20. We hypothesize that the induced ISG20 trims AGO- bound guide RNAs, thereby switching the main slicer from AGO2 to AGO3, to respond to the viral infection. In Aim 1, we will replace the nucleotides conserved among cityRNAs and validate their effect on target cleavage. To examine the involvement of AGO3-specific local structures in recognition of cityRNAs, we will make AGO3 mutants lacking either of the unique motifs and test them for in vitro target cleavage. We also will determine the crystal structures of AGO3 in complex with a 14-nt cityRNA to understand how AGO3 recognizes cityRNAs. The outcomes from Aim1 will provide the first insight into the requirements of cityRNA-loaded AGO3 for catalytic activation. In Aim 2, we will elucidate the mechanism of AGO3 activation. To determine the sequence complementarity between cityRNA and target RNAs required for target cleavage, we will systematically incorporate a single nucleotide mismatch at every position of the 14-nt miR-20a and let-7a. After loading with either of the cityRNAs, AGO3 will be tested for in vitro slicing activity. We will make target RNA variants and determine the minimum length of target RNA required for cityRNA-directed RNA cleavage. Lastly, we will determine the ternary complex crystal structures of AGO3 with the 14-nt miR-20a or let-7a and their target RNA. The outcomes from Aim2 will provide the molecular basis for target cleavage by cityRNA-loaded AGO3. In Aim 3, we will express FLAG-AGO2 or FLAG-AGO3 in HeLa cells with and without interferon treatment. Then, RNAs will be extracted from the immunopurified FLAG-AGO, followed by RNA sequencing. Using an approach that combines computational and methods, we will determine targets cleaved by AGO3-bound cityRNAs during innate immune activation in HeLa cells. The outcomes from Aim3 will identify the interferon-induced cityRNAs and their target RNAs. Altogeth... ## Key facts - **NIH application ID:** 10582158 - **Project number:** 3R01GM138997-03S1 - **Recipient organization:** OHIO STATE UNIVERSITY - **Principal Investigator:** Kotaro Nakanishi - **Activity code:** R01 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2022 - **Award amount:** $199,982 - **Award type:** 3 - **Project period:** 2020-07-10 → 2024-06-30 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10582158 ## Citation > US National Institutes of Health, RePORTER application 10582158, Structural and molecular basis for cityRNA (cleavage-inducing tiny RNA)-directed RNA cleavage by AGO3 (3R01GM138997-03S1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10582158. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*