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

Shintaro Iwasaki, Hiroshi M. Sasaki, Yuriko Sakaguchi, Tsutomu Suzuki, Hisashi Tadakuma () and Yukihide Tomari ()
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Shintaro Iwasaki: Institute of Molecular and Cellular Biosciences, The University of Tokyo
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

Nature, 2015, vol. 521, issue 7553, 533-536

Abstract: 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.

Date: 2015
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DOI: 10.1038/nature14254

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