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Defining fundamental steps in the assembly of the Drosophila RNAi enzyme complex

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  • Shintaro Iwasaki

    (Institute of Molecular and Cellular Biosciences, The University of Tokyo
    *Present address: Department of Molecular and Cell Biology, University of California, Berkeley, California, 94720, USA.)

  • Hiroshi M. Sasaki

    (Institute of Molecular and Cellular Biosciences, The University of Tokyo)

  • Yuriko Sakaguchi

    (Graduate School of Engineering, The University of Tokyo)

  • Tsutomu Suzuki

    (Graduate School of Engineering, The University of Tokyo)

  • Hisashi Tadakuma

    (Graduate School of Frontier Sciences, The University of Tokyo)

  • Yukihide Tomari

    (Institute of Molecular and Cellular Biosciences, The University of Tokyo
    Graduate School of Frontier Sciences, The University of Tokyo)

Abstract

Small RNAs such as small interfering RNAs (siRNAs) and microRNAs (miRNAs) silence the expression of their complementary target messenger RNAs1,2 via the formation of effector RNA-induced silencing complexes (RISCs), which contain Argonaute (Ago) family proteins at their core. Although loading of siRNA duplexes into Drosophila Ago2 requires the Dicer-2–R2D2 heterodimer3,4,5 and the Hsc70/Hsp90 (Hsp90 also known as Hsp83) chaperone machinery6,7,8, the details of RISC assembly remain unclear. Here we reconstitute RISC assembly using only Ago2, Dicer-2, R2D2, Hsc70, Hsp90, Hop, Droj2 (an Hsp40 homologue) and p23. By following the assembly of single RISC molecules, we find that, in the absence of the chaperone machinery, an siRNA bound to Dicer-2–R2D2 associates with Ago2 only transiently. The chaperone machinery extends the dwell time of the Dicer-2–R2D2–siRNA complex on Ago2, in a manner dependent on recognition of the 5′-phosphate on the siRNA guide strand. We propose that the chaperone machinery supports a productive state of Ago2, allowing it to load siRNA duplexes from Dicer-2–R2D2 and thereby assemble RISC.

Suggested Citation

  • Shintaro Iwasaki & Hiroshi M. Sasaki & Yuriko Sakaguchi & Tsutomu Suzuki & Hisashi Tadakuma & Yukihide Tomari, 2015. "Defining fundamental steps in the assembly of the Drosophila RNAi enzyme complex," Nature, Nature, vol. 521(7553), pages 533-536, May.
    • Handle: RePEc:nat:nature:v:521:y:2015:i:7553:d:10.1038_nature14254
    DOI: 10.1038/nature14254
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