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Structural determinants of miRNAs for RISC loading and slicer-independent unwinding

Nature Structural & Molecular Biology volume 16, pages 953–960 (2009)Cite this article

Abstract

MicroRNAs (miRNAs) regulate expression of their target mRNAs through the RNA-induced silencing complex (RISC), which contains an Argonaute (Ago) family protein as a core component. In Drosophila melanogaster, miRNAs are generally sorted into Ago1-containing RISC (Ago1-RISC). We established a native gel system that can biochemically dissect the Ago1-RISC assembly pathway. We found that miRNA-miRNA* duplexes are loaded into Ago1 as double-stranded RNAs in an ATP-dependent fashion. In contrast, unexpectedly, unwinding of miRNA-miRNA* duplexes is a passive process that does not require ATP or slicer activity of Ago1. Central mismatches direct miRNA-miRNA* duplexes into pre-Ago1-RISC, whereas mismatches in the seed or guide strand positions 12–15 promote conversion of pre-Ago1-RISC into mature Ago1-RISC. Our findings show that unwinding of miRNAs is a precise mirror-image process of target recognition, and both processes reflect the unique geometry of RNAs in Ago proteins.

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Figure 1: Identification of small RNA–protein complexes in dcr-2 embryo lysate.
Figure 2: Complex I is pre-Ago1-RISC, and complex II is mature Ago1-RISC.
Figure 3: ATP is essential for Ago1-RISC loading but dispensable for unwinding.
Figure 4: Central mismatches direct small RNA duplexes into pre-Ago1-RISC, whereas seed or 3′-mid mismatches promote conversion of pre-Ago1-RISC to mature Ago1-RISC.
Figure 5: Strand separation of small RNA duplex does not require the slicer activity of Ago1.
Figure 6: Structural determinants for Ago1-RISC loading and strand separation.

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Acknowledgements

We thank M. Siomi and H. Siomi (Keio University) for the antibody to Ago1, E. Izaurralde (Max Planck Institute) for the antibody to GW182, R. Carthew (Northwestern University) for the dcr-2L811fsX flies, and M. Horwich and P. Zamore (University of Massachusetts) for the pENTR-Ago1 construct. We also thank M. Mitomi and A. Maruyama for technical assistance, S. Iwasaki and members of the Tomari laboratory for constant and helpful discussions, and H. Sasaki and P. Zamore for stimulating discussions. This work was supported in part by a Grant-in-Aid for Young Scientists (A) from the Japan Ministry of Education, Culture, Sports, Science and Technology, and a Carrier Development Award from The International Human Frontier Science Program Organization. Y.T. is a Japan Science and Technology Agency PRESTO researcher.

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Authors and Affiliations

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

    Tomoko Kawamata & Yukihide Tomari

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

    Tomoko Kawamata & Yukihide Tomari

  3. Université de Toulouse, UPS, Laboratoire de Biologie Moléculaire Eucaryote, Toulouse, France

    Hervé Seitz

  4. CNRS, Laboratoire de Biologie Moléculaire Eucaryote, Toulouse, France

    Hervé Seitz

  5. PRESTO, Japan Science and Technology Agency, Kawaguchi-shi, Saitama, Japan

    Yukihide Tomari

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  1. Tomoko Kawamata
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T.K. and Y.T. conducted biochemical experiments, H.S. conducted bioinformatic analyses, Y.T. supervised the study, and all authors discussed the results and wrote the manuscript.

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Correspondence to Yukihide Tomari.

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Kawamata, T., Seitz, H. & Tomari, Y. Structural determinants of miRNAs for RISC loading and slicer-independent unwinding. Nat Struct Mol Biol 16, 953–960 (2009). https://doi.org/10.1038/nsmb.1630

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