Poster abstracts

Poster number 158 submitted by Huaqun Zhang

Gene silencing by cityRNAs

Huaqun Zhang (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio, 43210, USA), GeunYoung Sim (Molecular, Cellular and Developmental Biology, Center for RNA Biology, The Ohio State University, Columbus, Ohio, 43210, USA), Audrey C. Kehling, Vishal Annasaheb Adhav (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio, 43210, USA), Andrew Savidge (Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio, 43210, USA), Benjamin Pastore, Wen Tang (Center for RNA Biology, Ohio State Biochemistry Program, Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, Ohio, 43210, USA), Kotaro Nakanishi (Department of Chemistry and Biochemistry, Molecular, Cellular and Developmental Biology, Center for RNA Biology, Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio, 43210, USA)

Abstract:
Huaqun Zhang, GeunYoung Sim, and Audrey C. Kehling contributed equally.

Argonaute proteins (AGOs) load 20-23 nucleotide (nt) microRNAs (miRNAs) to form the RNA-induced silencing complex (RISC), the effector of RNA interference. Among the four human AGO paralogs, only AGO2 and AGO3 cleave target RNAs that are fully complementary to the guide RNA1. Recently, we discovered that AGO3 maximizes its endoribonuclease activity by loading specific guide RNAs of approximately 14 nt in length. These newly identified small noncoding RNAs have been termed cleavage-inducing tiny guide RNAs (cityRNAs)2. In this study, our in vitro assays demonstrated that the upstream sequence of the tiny RNA binding site (UTy) on the target RNA is crucial for efficient cleavage by AGO3, with a similar trend observed in AGO23. We further evaluated the potential of cityRNAs as a gene-silencing tool. To this end, we developed two auxiliary RNA systems, referred to as Booster, which facilitate the loading of any desired cityRNAs into endogenous AGO proteins. Using Dual-Luciferase Reporter assays, we demonstrated that cityRNAs efficiently induced gene silencing in HEK293T, HCT116, A549, and HeLa cells, especially for targets with specific UTy sequences. Unexpectedly, our data suggest that cityRNA-mediated silencing is highly dependent on target RNA cleavage. This could potentially reduce the off-target effect that designed small interfering RNAs bind to undesired target RNAs and induce silencing via translational repression.

References:
1. Park, M.S., Phan, H.D., Busch, F., Hinckley, S.H., Brackbill, J.A., Wysocki, V.H., and Nakanishi, K. (2017). Human Argonaute3 has slicer activity. Nucleic Acids Res 45, 11867-11877. 10.1093/nar/gkx916.
2. Park, M.S., Sim, G., Kehling, A.C., and Nakanishi, K. (2020). Human Argonaute2 and Argonaute3 are catalytically activated by different lengths of guide RNA. Proc Natl Acad Sci U S A 117, 28576-28578. 10.1073/pnas.2015026117.
3. Zhang, H., Sim, G.Y., Kehling, A.C., Adhav, V.A., Savidge, A., Pastore, B., Tang, W., Nakanishi, K. Cell Rep. accepted

Keywords: Argonaute, cityRNA, gene silencing