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Nature. 2015 May 28;521(7553):533-6. doi: 10.1038/nature14254. Epub 2015 Mar 30.

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

Author information

1
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan.
2
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
3
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan.
4
1] Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan. [2] Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan.

Abstract

Small RNAs such as small interfering RNAs (siRNAs) and microRNAs (miRNAs) silence the expression of their complementary target messenger RNAs 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 heterodimer and the Hsc70/Hsp90 (Hsp90 also known as Hsp83) chaperone machinery, 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.

PMID:
25822791
DOI:
10.1038/nature14254
[Indexed for MEDLINE]

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