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Title: Structural and functional analyses reveal the contributions of the C- and N-lobes of Argonaute protein to selectivity of RNA target cleavage

Abstract

Some gene transcripts have cellular functions as regulatory noncoding RNAs. For example, ~23-nucleotide (nt)–long siRNAs are loaded into Argonaute proteins. The resultant ribonucleoprotein assembly, the RNA-induced silencing complex (RISC), cleaves RNAs that are extensively base-paired with the loaded siRNA. To date, base complementarity is recognized as the major determinant of specific target cleavage (or slicing), but little is known about how Argonaute inspects base pairing before cleavage. A hallmark of Argonaute proteins is their bilobal structure, but despite the significance of this structure for curtailing slicing activity against mismatched targets, the molecular mechanism remains elusive. Here, our structural and functional studies of a bilobed yeast Argonaute protein and its isolated catalytic C-terminal lobe (C-lobe) revealed that the C-lobe alone retains almost all properties of bilobed Argonaute: siRNA-duplex loading, passenger cleavage/ejection, and siRNAdependent RNA cleavage. A 2.1 Å–resolution crystal structure revealed that the catalytic C-lobe mirrors the bilobed Argonaute in terms of guide-RNA recognition and that all requirements for transitioning to the catalytically active conformation reside in the C-lobe. Nevertheless, we found that in the absence of the N-terminal lobe (N-lobe), target RNAs are scanned for complementarity only at positions 5–14 on a 23-nt guide RNA before endonucleolytic cleavage, thereby allowingmore » for some off-target cleavage. Of note, acquisition of an N-lobe expanded the range of the guide RNA strand used for inspecting target complementarity to positions 2–23. To summarize, these findings offer clues to the evolution of the bilobal structure of catalytically active Argonaute proteins.« less

Authors:
 [1];  [1];  [1]
  1. Ohio State Univ., Columbus, OH (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
Center for RNA Biology Graduate Student Fellowship; Pelotonia Graduate Student Cancer Research Fellowship; Ohio State University Start-up; Ohio State University Center for RNA Biology Seed Grant; National Institutes of Health (NIH); USDOE Office of Science (SC)
OSTI Identifier:
1435839
Grant/Contract Number:  
GUP-41799; R01GM124320; S10OD023582; P41 GM103403; S10OD021527; AC02–06CH11357
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 293; Journal Issue: 17; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; RNA Argonaute; X-ray crystallography; ribonuclear protein (RNP); structural biology

Citation Formats

Dayeh, Daniel M., Kruithoff, Bradley C., and Nakanishi, Kotaro. Structural and functional analyses reveal the contributions of the C- and N-lobes of Argonaute protein to selectivity of RNA target cleavage. United States: N. p., 2018. Web. doi:10.1074/jbc.RA117.001051.
Dayeh, Daniel M., Kruithoff, Bradley C., & Nakanishi, Kotaro. Structural and functional analyses reveal the contributions of the C- and N-lobes of Argonaute protein to selectivity of RNA target cleavage. United States. doi:10.1074/jbc.RA117.001051.
Dayeh, Daniel M., Kruithoff, Bradley C., and Nakanishi, Kotaro. Thu . "Structural and functional analyses reveal the contributions of the C- and N-lobes of Argonaute protein to selectivity of RNA target cleavage". United States. doi:10.1074/jbc.RA117.001051. https://www.osti.gov/servlets/purl/1435839.
@article{osti_1435839,
title = {Structural and functional analyses reveal the contributions of the C- and N-lobes of Argonaute protein to selectivity of RNA target cleavage},
author = {Dayeh, Daniel M. and Kruithoff, Bradley C. and Nakanishi, Kotaro},
abstractNote = {Some gene transcripts have cellular functions as regulatory noncoding RNAs. For example, ~23-nucleotide (nt)–long siRNAs are loaded into Argonaute proteins. The resultant ribonucleoprotein assembly, the RNA-induced silencing complex (RISC), cleaves RNAs that are extensively base-paired with the loaded siRNA. To date, base complementarity is recognized as the major determinant of specific target cleavage (or slicing), but little is known about how Argonaute inspects base pairing before cleavage. A hallmark of Argonaute proteins is their bilobal structure, but despite the significance of this structure for curtailing slicing activity against mismatched targets, the molecular mechanism remains elusive. Here, our structural and functional studies of a bilobed yeast Argonaute protein and its isolated catalytic C-terminal lobe (C-lobe) revealed that the C-lobe alone retains almost all properties of bilobed Argonaute: siRNA-duplex loading, passenger cleavage/ejection, and siRNAdependent RNA cleavage. A 2.1 Å–resolution crystal structure revealed that the catalytic C-lobe mirrors the bilobed Argonaute in terms of guide-RNA recognition and that all requirements for transitioning to the catalytically active conformation reside in the C-lobe. Nevertheless, we found that in the absence of the N-terminal lobe (N-lobe), target RNAs are scanned for complementarity only at positions 5–14 on a 23-nt guide RNA before endonucleolytic cleavage, thereby allowing for some off-target cleavage. Of note, acquisition of an N-lobe expanded the range of the guide RNA strand used for inspecting target complementarity to positions 2–23. To summarize, these findings offer clues to the evolution of the bilobal structure of catalytically active Argonaute proteins.},
doi = {10.1074/jbc.RA117.001051},
journal = {Journal of Biological Chemistry},
issn = {0021-9258},
number = 17,
volume = 293,
place = {United States},
year = {2018},
month = {3}
}

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