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Programmable RNA targeting by bacterial Argonaute nucleases with unconventional guide binding and cleavage specificity

Lisitskaya, Lidiya and Shin, Yeonoh and Agapov, Aleksei and Olina, Anna and Kropocheva, Ekaterina and Ryazansky, Sergei and Aravin, Alexei A. and Esyunina, Daria and Murakami, Katsuhiko S. and Kulbachinskiy, Andrey (2022) Programmable RNA targeting by bacterial Argonaute nucleases with unconventional guide binding and cleavage specificity. Nature Communications, 13 . Art. No. 4624. ISSN 2041-1723. PMCID PMC9360449. doi:10.1038/s41467-022-32079-5.

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Argonaute proteins are programmable nucleases that have defense and regulatory functions in both eukaryotes and prokaryotes. All known prokaryotic Argonautes (pAgos) characterized so far act on DNA targets. Here, we describe a new class of pAgos that uniquely use DNA guides to process RNA targets. The biochemical and structural analysis of Pseudooceanicola lipolyticus pAgo (PliAgo) reveals an unusual organization of the guide binding pocket that does not rely on divalent cations and the canonical set of contacts for 5’-end interactions. Unconventional interactions of PliAgo with the 5’-phosphate of guide DNA define its new position within pAgo and shift the site of target RNA cleavage in comparison with known Argonautes. The specificity for RNA over DNA is defined by ribonucleotide residues at the cleavage site. The analysed pAgos sense mismatches and modifications in the RNA target. The results broaden our understanding of prokaryotic defense systems and extend the spectrum of programmable nucleases with potential use in RNA technology.

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URLURL TypeDescription CentralArticle
Lisitskaya, Lidiya0000-0002-5283-7983
Shin, Yeonoh0000-0001-9718-2995
Agapov, Aleksei0000-0002-8662-8573
Olina, Anna0000-0003-1844-2109
Ryazansky, Sergei0000-0002-3171-2997
Aravin, Alexei A.0000-0002-6956-8257
Esyunina, Daria0000-0002-3706-4425
Murakami, Katsuhiko S.0000-0003-2244-0501
Kulbachinskiy, Andrey0000-0002-2292-6424
Additional Information:© The Author(s) 2022. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit Received 14 June 2022. Accepted 15 July 2022. Published 08 August 2022. We thank the X-ray crystallography facility at Pennsylvania State University for screening crystallization conditions and Macromolecular X-ray science staff at the Cornell High Energy Synchrotron Source (MacCHESS) for supporting the crystallographic data collection, Dr. Alexandra Kulikova and Dr. Vladimir Mitkevich for analysis of protein thermal stability, Dr. Mayya Petrova for help with bacterial strains, Dr. Alexei Kotov for help with confocal microscopy. This study was supported in part by the Russian Science Foundation (grant 20-74-10127 to A. Agapov, analysis of nucleic acid specificity of pAgos; grant 22-14-00182 to A.K., analysis of mutant variants of pAgos) and National Institutes of Health (R35 GM131860 to KSM, structural analysis). Contributions. A.A.A., D.E., K.S.M., and A.K. conceptualized the study. L.L., A.A., A.O., and E.K. designed and performed biochemical experiments, Y.S. performed crystallization and X-ray diffraction studies, Y.S. and K.S.M. performed structural analysis, S.R. performed bioinformatic analysis, A.K. and K.S.M. wrote the manuscript with input from all coauthors. Data availability. The data that support this study are available from the corresponding authors upon request. The coordinates are deposited in the Protein Data Bank with PDB accession codes 7R8F (native PliAgo), 7R8G (PliAgo-OH-gDNA complex), 7R8H (PliAgo-P-gDNA complex), 7R8J (PliAgo-P-gDNA-Mg2+ complex) and 7R8K (SeMet PliAgo). Source data are provided with this paper. The authors declare no competing interests. Peer review information. Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.
Funding AgencyGrant Number
Russian Science Foundation20-74-10127
Russian Science Foundation22-14-00182
NIHR35 GM131860
PubMed Central ID:PMC9360449
Record Number:CaltechAUTHORS:20220816-373347000
Persistent URL:
Official Citation:Lisitskaya, L., Shin, Y., Agapov, A. et al. Programmable RNA targeting by bacterial Argonaute nucleases with unconventional guide binding and cleavage specificity. Nat Commun 13, 4624 (2022).
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:116302
Deposited By: George Porter
Deposited On:16 Aug 2022 20:06
Last Modified:16 Aug 2022 20:06

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