Single-molecule FRET Uncovers Hidden Conformations and Dynamics of Human Argonaute 2

Overview

Journal Nat Commun

Specialty Biology

Date 2022 Jul 2

PMID 35780145

Authors

Sarah Willkomm

Leonhard Jakob

Kevin Kramm

Veronika Graus

Julia Neumeier

Gunter Meister

Dina Grohmann

Affiliations

Soon will be listed here.

Abstract

Human Argonaute 2 (hAgo2) constitutes the functional core of the RNA interference pathway. Guide RNAs direct hAgo2 to target mRNAs, which ultimately leads to hAgo2-mediated mRNA degradation or translational inhibition. Here, we combine site-specifically labeled hAgo2 with time-resolved single-molecule FRET measurements to monitor conformational states and dynamics of hAgo2 and hAgo2-RNA complexes in solution that remained elusive so far. We observe dynamic anchoring and release of the guide's 3'-end from the PAZ domain during the stepwise target loading process even with a fully complementary target. We find differences in structure and dynamic behavior between partially and fully paired canonical hAgo2-guide/target complexes and the miRNA processing complex formed by hAgo2 and pre-miRNA451. Furthermore, we detect a hitherto unknown conformation of hAgo2-guide/target complexes that poises them for target-directed miRNA degradation. Taken together, our results show how the conformational flexibility of hAgo2-RNA complexes determines function and the fate of the ribonucleoprotein particle.

Citing Articles

The structural basis for RNA slicing by human Argonaute2.

Mohamed A, Wang P, Bartel D, Vos S Cell Rep. 2025; 44(1):115166.

PMID: 39932188 PMC: 11893014. DOI: 10.1016/j.celrep.2024.115166.

Importance of an N-terminal structural switch in the distinction between small RNA-bound and free ARGONAUTE.

Bressendorff S, Sjogaard I, Prestel A, Voutsinos V, Jansson M, Menard P Nat Struct Mol Biol. 2025; .

PMID: 39774835 DOI: 10.1038/s41594-024-01446-9.

Structural basis for gene silencing by siRNAs in humans.

Sarkar S, Gebert L, MacRae I bioRxiv. 2024; .

PMID: 39677650 PMC: 11643337. DOI: 10.1101/2024.12.05.627081.

Exploring the dynamics of messenger ribonucleoprotein-mediated translation repression.

Meyer J, Payr M, Duss O, Hennig J Biochem Soc Trans. 2024; 52(6):2267-2279.

PMID: 39601754 PMC: 11668304. DOI: 10.1042/BST20231240.

The structural basis for RNA slicing by human Argonaute2.

Mohamed A, Wang P, Bartel D, Vos S bioRxiv. 2024; .

PMID: 39229170 PMC: 11370433. DOI: 10.1101/2024.08.19.608718.

References

Tomari Y, Zamore P . Perspective: machines for RNAi. Genes Dev. 2005; 19(5):517-29. DOI: 10.1101/gad.1284105. View

Tsuboyama K, Tadakuma H, Tomari Y . Conformational Activation of Argonaute by Distinct yet Coordinated Actions of the Hsp70 and Hsp90 Chaperone Systems. Mol Cell. 2018; 70(4):722-729.e4. DOI: 10.1016/j.molcel.2018.04.010. View

Martinez J, Tuschl T . RISC is a 5' phosphomonoester-producing RNA endonuclease. Genes Dev. 2004; 18(9):975-80. PMC: 406288. DOI: 10.1101/gad.1187904. View

Preus S, Noer S, Hildebrandt L, Gudnason D, Birkedal V . iSMS: single-molecule FRET microscopy software. Nat Methods. 2015; 12(7):593-4. DOI: 10.1038/nmeth.3435. View

Jo M, Shin S, Jung S, Kim E, Song J, Hohng S . Human Argonaute 2 Has Diverse Reaction Pathways on Target RNAs. Mol Cell. 2015; 59(1):117-24. DOI: 10.1016/j.molcel.2015.04.027. View

Sheu-Gruttadauria J, Pawlica P, Klum S, Wang S, Yario T, Schirle Oakdale N . Structural Basis for Target-Directed MicroRNA Degradation. Mol Cell. 2019; 75(6):1243-1255.e7. PMC: 6754277. DOI: 10.1016/j.molcel.2019.06.019. View

Willkomm S, Oellig C, Zander A, Restle T, Keegan R, Grohmann D . Structural and mechanistic insights into an archaeal DNA-guided Argonaute protein. Nat Microbiol. 2017; 2:17035. DOI: 10.1038/nmicrobiol.2017.35. View

Wang Y, Juranek S, Li H, Sheng G, Tuschl T, Patel D . Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex. Nature. 2008; 456(7224):921-6. PMC: 2765400. DOI: 10.1038/nature07666. View

Dykxhoorn D, Novina C, Sharp P . Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol. 2003; 4(6):457-67. DOI: 10.1038/nrm1129. View

10.

Krol J, Busskamp V, Markiewicz I, Stadler M, Ribi S, Richter J . Characterizing light-regulated retinal microRNAs reveals rapid turnover as a common property of neuronal microRNAs. Cell. 2010; 141(4):618-31. DOI: 10.1016/j.cell.2010.03.039. View

11.

Liu J, Carmell M, Rivas F, Marsden C, Thomson J, Song J . Argonaute2 is the catalytic engine of mammalian RNAi. Science. 2004; 305(5689):1437-41. DOI: 10.1126/science.1102513. View

12.

MacRae I, Ma E, Zhou M, Robinson C, Doudna J . In vitro reconstitution of the human RISC-loading complex. Proc Natl Acad Sci U S A. 2008; 105(2):512-7. PMC: 2206567. DOI: 10.1073/pnas.0710869105. View

13.

Bronson J, Fei J, Hofman J, Gonzalez Jr R, Wiggins C . Learning rates and states from biophysical time series: a Bayesian approach to model selection and single-molecule FRET data. Biophys J. 2009; 97(12):3196-205. PMC: 2793368. DOI: 10.1016/j.bpj.2009.09.031. View

14.

Jung S, Kim E, Hwang W, Shin S, Song J, Hohng S . Dynamic anchoring of the 3'-end of the guide strand controls the target dissociation of Argonaute-guide complex. J Am Chem Soc. 2013; 135(45):16865-71. DOI: 10.1021/ja403138d. View

15.

Moore M, Scheel T, Luna J, Park C, Fak J, Nishiuchi E . miRNA-target chimeras reveal miRNA 3'-end pairing as a major determinant of Argonaute target specificity. Nat Commun. 2015; 6:8864. PMC: 4674787. DOI: 10.1038/ncomms9864. View

16.

Meister G, Landthaler M, Patkaniowska A, Dorsett Y, Teng G, Tuschl T . Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol Cell. 2004; 15(2):185-97. DOI: 10.1016/j.molcel.2004.07.007. View

17.

Chandradoss S, Schirle N, Szczepaniak M, MacRae I, Joo C . A Dynamic Search Process Underlies MicroRNA Targeting. Cell. 2015; 162(1):96-107. PMC: 4768356. DOI: 10.1016/j.cell.2015.06.032. View

18.

Rissland O, Hong S, Bartel D . MicroRNA destabilization enables dynamic regulation of the miR-16 family in response to cell-cycle changes. Mol Cell. 2011; 43(6):993-1004. PMC: 3202612. DOI: 10.1016/j.molcel.2011.08.021. View

19.

Kwak P, Tomari Y . The N domain of Argonaute drives duplex unwinding during RISC assembly. Nat Struct Mol Biol. 2012; 19(2):145-51. DOI: 10.1038/nsmb.2232. View

20.

Wee L, Flores-Jasso C, Salomon W, Zamore P . Argonaute divides its RNA guide into domains with distinct functions and RNA-binding properties. Cell. 2012; 151(5):1055-67. PMC: 3595543. DOI: 10.1016/j.cell.2012.10.036. View