Target cleavage and gene silencing by Argonautes with cityRNAs
Huaqun Zhang,
GeunYoung Sim,
Audrey C. Kehling,
Vishal Annasaheb Adhav,
Andrew Savidge,
Benjamin Pastore,
Wen Tang,
Kotaro Nakanishi
Affiliations
Huaqun Zhang
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA
GeunYoung Sim
Molecular, Cellular and Developmental Biology, The Ohio State University, Columbus, OH 43210, USA; Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA
Audrey C. Kehling
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA
Vishal Annasaheb Adhav
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA
Andrew Savidge
Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA
Benjamin Pastore
Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA; Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA; Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, OH 43210, USA
Wen Tang
Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA; Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA; Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, OH 43210, USA
Kotaro Nakanishi
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA; Molecular, Cellular and Developmental Biology, The Ohio State University, Columbus, OH 43210, USA; Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA; Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA; Corresponding author
Summary: TinyRNAs (tyRNAs) are ≤17-nt guide RNAs associated with Argonaute proteins (AGOs), and certain 14-nt cleavage-inducing tyRNAs (cityRNAs) catalytically activate human Argonaute3 (AGO3). We present the crystal structure of AGO3 in complex with a cityRNA, 14-nt miR-20a, and its complementary target, revealing a different trajectory for the guide-target duplex from that of its ∼22-nt microRNA-associated AGO counterpart. cityRNA-loaded Argonaute2 (AGO2) and AGO3 enhance their endonuclease activity when the immediate 5′ upstream region of the tyRNA target site (UTy) includes sequences with low affinity for AGO. We propose a model where cityRNA-loaded AGO2 and AGO3 efficiently cleave fully complementary tyRNA target sites unless they directly recognize the UTy. To investigate their gene silencing, we devised systems for loading endogenous AGOs with specific tyRNAs and demonstrated that, unlike microRNAs, cityRNA-mediated silencing heavily relies on target cleavage. Our study uncovered that AGO exploits cityRNAs for target recognition differently from microRNAs and alters gene silencing.
