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Accommodation of Helical Imperfections in Rhodobacter sphaeroides Argonaute Ternary Complexes with Guide RNA and Target DNA

Liu, Yiwei and Esyunina, Daria and Olovnikov, Ivan and Teplova, Marianna and Kulbachinskiy, Andrey and Aravin, Alexei A. and Patel, Dinshaw J. (2018) Accommodation of Helical Imperfections in Rhodobacter sphaeroides Argonaute Ternary Complexes with Guide RNA and Target DNA. Cell Reports, 24 (2). pp. 453-462. ISSN 2211-1247. PMCID PMC6269105. http://resolver.caltech.edu/CaltechAUTHORS:20180711-124213871

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Abstract

Prokaryotic Argonaute (Ago) proteins were recently shown to target foreign genetic elements, thus making them a perfect model for studies of interference mechanisms. Here, we study interactions of Rhodobacter sphaeroides Ago (RsAgo) with guide RNA (gRNA) and fully complementary or imperfect target DNA (tDNA) using biochemical and structural approaches. We show that RsAgo can specifically recognize both the first nucleotide in gRNA and complementary nucleotide in tDNA, and both interactions contribute to nucleic acid binding. Non-canonical pairs and bulges on the target strand can be accommodated by RsAgo with minimal perturbation of the duplex but significantly reduce RsAgo affinity to tDNA. Surprisingly, mismatches between gRNA and tDNA induce dissociation of the guide-target duplex from RsAgo. Our results reveal plasticity in the ability of Ago proteins to accommodate helical imperfections, show how this might affect the efficiency of RNA silencing, and suggest a potential mechanism for guide release and Ago recycling.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/j.celrep.2018.06.021DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6269105PubMed CentralArticle
Additional Information:© 2018 The Authors. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Received 1 August 2017, Revised 11 April 2018, Accepted 5 June 2018, Available online 11 July 2018. Open Access funded by National Institutes of Health. We thank the synchrotron beamline staff at the Argonne National Laboratory for their assistance. D.J.P. was supported by NIH Grant TR01 GM104962; A.A.A. was supported by NIH Grants R01 GM097369, R00 HD057233, and DP2 OD007371A and a Searle Scholar Award; and D.E. and A.K. were supported by Russian Science Foundation Grant 16-14-10377. D.J.P. was supported by NIH Grant GM104962 and the Memorial Sloan Kettering Cancer Center Core Grant (P30 CA008748). Author Contributions: X-ray structure determination of RsAgo ternary complexes was performed by Y.L. and M.T. under the supervision of D.J.P. The identification of RsAgo specificity for gRNA and tDNA was performed by I.O. under the supervision of A.A.A. Analysis of interactions of RsAgo with guide and target molecules was performed by D.E. under the supervision of A.K. and A.A.A. The authors declare no competing interests. Data and Software Availability: The accession numbers for the ternary RsAgo complexes reported in this study are PDB: 6D8P (ternary RsAgo complex with fully paired gRNA-target DNA duplex), 6D92 (ternary RsAgo complex containing A3⋅A3′ pair), 6D95 (ternary RsAgo complex containing A8⋅A8′ pair), 6D9K (ternary RsAgo complex containing A8⋅G8′ pair), 6D9L (ternary RsAgo complex containing G8⋅A8′ pair), 6D8A (ternary RsAgo complex containing A-A bulge), and 6D8F (ternary RsAgo complex containing T-T bulge).
Funders:
Funding AgencyGrant Number
NIHTR01 GM104962
NIHR01 GM097369
NIHR00 HD057233
NIHDP2 OD007371A
Searle Scholars ProgramUNSPECIFIED
Russian Science Foundation16-14-10377
NIHGM104962
NIHP30 CA008748
Subject Keywords:Rhodobacter sphaeroides Argonaute; RsAgo; guide RNA; target DNA; RNA-DNA heteroduplex; non-canonical base pairs and bulges
PubMed Central ID:PMC6269105
Record Number:CaltechAUTHORS:20180711-124213871
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20180711-124213871
Official Citation:Yiwei Liu, Daria Esyunina, Ivan Olovnikov, Marianna Teplova, Andrey Kulbachinskiy, Alexei A. Aravin, Dinshaw J. Patel, Accommodation of Helical Imperfections in Rhodobacter sphaeroides Argonaute Ternary Complexes with Guide RNA and Target DNA, Cell Reports, Volume 24, Issue 2, 2018, Pages 453-462, ISSN 2211-1247, https://doi.org/10.1016/j.celrep.2018.06.021. (http://www.sciencedirect.com/science/article/pii/S2211124718309161)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:87760
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:11 Jul 2018 19:57
Last Modified:03 Dec 2018 15:41

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