Article | Published:

The N domain of Argonaute drives duplex unwinding during RISC assembly

Nature Structural & Molecular Biology volume 19, pages 145151 (2012) | Download Citation

Abstract

Small RNAs, such as microRNAs and small interfering RNAs, act through Argonaute (Ago) proteins as a part of RNA-induced silencing complexes (RISCs). To make RISCs, Ago proteins bind and subsequently unwind small RNA duplexes, finally leaving one strand stably incorporated. Here we identified the N domain of human AGO2 as the initiator of duplex unwinding during RISC assembly. We discovered that a functional N domain is strictly required for small RNA duplex unwinding but not for precedent duplex loading or subsequent target cleavage. We postulate that RISC assembly is tripartite, comprising (i) RISC loading, whereby Ago undergoes conformational opening and loads a small RNA duplex, forming pre-RISC; (ii) wedging, whereby the end of the duplex is pried open through active wedging by the N domain, in preparation for unwinding; and (iii) unwinding, whereby the passenger strand is removed through slicer-dependent or slicer-independent unwinding, forming mature RISC.

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Acknowledgements

We acknowledge T. Tuschl (Rockefeller University) for the pIRESneo-Flag-hemagglutinin-AGO2 plasmid and S. Katsuma (The University of Tokyo) for technical assistance. We are grateful to H. Seitz, T. Kawamata and members of the Tomari laboratory for helpful discussions and suggestions and critical comments on the manuscript. This work was supported in part by a Grant-in-Aid for Scientific Research on Innovative Areas (“Functional machinery for non-coding RNAs”) to Y.T. P.B.K. is a Prins Bernhard Cultuurfonds Fellow, supported by the Banning-de Jong Fonds.

Author information

Affiliations

  1. Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

    • Pieter Bas Kwak
    •  & Yukihide Tomari
  2. Department of Medical Genome Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

    • Pieter Bas Kwak
    •  & Yukihide Tomari

Authors

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Contributions

P.B.K. conducted all experiments, Y.T. supervised the study, and P.B.K. and Y.T. wrote the manuscript, discussed the results and approved the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Yukihide Tomari.

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DOI

https://doi.org/10.1038/nsmb.2232

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