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ATP-dependent human RISC assembly pathways

Nature Structural & Molecular Biology volume 17, pages 17–23 (2010)Cite this article

Abstract

The assembly of RNA-induced silencing complex (RISC) is a key process in small RNA–mediated gene silencing. In humans, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are incorporated into RISCs containing the Argonaute (AGO) subfamily proteins Ago1–4. Previous studies have proposed that, unlike Drosophila melanogaster RISC assembly pathways, human RISC assembly is coupled with dicing and is independent of ATP. Here we show by careful reexamination that, in humans, RISC assembly and dicing are uncoupled, and ATP greatly facilitates RISC loading of small-RNA duplexes. Moreover, all four human AGO proteins show remarkably similar structural preferences for small-RNA duplexes: central mismatches promote RISC loading, and seed or 3′-mid (guide position 12–15) mismatches facilitate unwinding. All these features of human AGO proteins are highly reminiscent of fly Ago1 but not fly Ago2.

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Figure 1: Human RISC assembly is uncoupled from dicing and dependent on ATP.
Figure 2: Complex I and complex II are pre–Ago2 RISC and mature–Ago2 RISC, respectively.
Figure 3: ATP facilitates RISC loading but not unwinding.
Figure 4: Central mismatches promote Ago2-RISC loading, and seed and 3′-mid mismatches facilitate unwinding.
Figure 5: Four human AGO proteins have similar structural preferences for small-RNA duplexes for RISC loading and unwinding.
Figure 6: Ago1–4 can use miRNA-like duplexes, whereas only Ago2 can efficiently unwind siRNA duplexes.

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Acknowledgements

We acknowledge T. Tuschl (Rockefeller University) for pIRESneo-Flag-HA-Ago1–4 plasmids, C. Cepko (Harvard Medical School) for pCAGEN vector and M. and H. Siomi (Keio University) for anti–human Ago2. We are also grateful to T. Katoh, T. Suzuki and K. Ui-Tei for helpful discussion, T. Okazaki for his extensive technical assistance and A. Nomoto for his generous support and encouragement. We thank the members of the Tomari laboratory, S. Kawaoka, H. Sasaki, K. Förstemann and H. Seitz for suggestions and critical comments on the manuscript. This work was supported in part by a Grant-in-Aid for Young Scientists (A) to Y.T. and a Grant-in-Aid for Young Scientists (B) to T.K. from the Japan Ministry of Education, Culture, Sports, Science and Technology, a grant from the Welch foundation to Q.L. and a Carrier Development Award from The International Human Frontier Science Program Organization to Y.T. Y.T. is a Japan Science and Technology Agency Precursory Research for Embryonic Science and Technology researcher.

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Authors and Affiliations

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

    Mayuko Yoda, Tomoko Kawamata, Shintaro Iwasaki & Yukihide Tomari

  2. Department of Medical Genome Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.,

    Mayuko Yoda, Tomoko Kawamata, Shintaro Iwasaki & Yukihide Tomari

  3. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Zain Paroo, Xuecheng Ye & Qinghua Liu

  4. Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi-shi, Saitama, Japan.,

    Yukihide Tomari

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  1. Mayuko Yoda
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Contributions

M.Y. performed in vivo experiments; M.Y., T.K. and S.I. performed biochemical experiments; Z.P., X.Y. and Q.L. expressed and purified recombinant Ago2 and performed initial experiments using it; Y.T. supervised the study; M.Y. and Y.T. wrote the manuscript; all authors discussed the results and approved the manuscript.

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Correspondence to Yukihide Tomari.

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Yoda, M., Kawamata, T., Paroo, Z. et al. ATP-dependent human RISC assembly pathways. Nat Struct Mol Biol 17, 17–23 (2010). https://doi.org/10.1038/nsmb.1733

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