In vitro reconstitution of chaperone-mediated human RISC assembly
- Ken Naruse1,2,3,6,
- Eriko Matsuura-Suzuki1,6,
- Mariko Watanabe1,2,
- Shintaro Iwasaki1,4,5 and
- Yukihide Tomari1,2
- 1Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan
- 2Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan
- 3Department of Biology, Graduate School of Science, Kobe University, Kobe 657-8501, Japan
- 4Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Wako, Saitama 351-0198, Japan
- 5RNA Systems Biochemistry Laboratory, RIKEN, Wako, Saitama 351-0198, Japan
- Corresponding authors: shintaro.iwasaki{at}riken.jp, tomari{at}iam.u-tokyo.ac.jp
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↵6 These authors contributed equally to this work.
Abstract
To silence target mRNAs, small RNAs and Argonaute (Ago) proteins need to be assembled into RNA-induced silencing complexes (RISCs). Although the assembly of Drosophila melanogaster RISC was recently reconstituted by Ago2, the Dicer-2/R2D2 heterodimer, and five chaperone proteins, the absence of a reconstitution system for mammalian RISC assembly has posed analytical challenges. Here we describe reconstitution of human RISC assembly using Ago2 and five recombinant chaperone proteins: Hsp90β, Hsc70, Hop, Dnaja2, and p23. Our data show that ATP hydrolysis by both Hsp90β and Hsc70 is required for RISC assembly of small RNA duplexes but not for that of single-stranded RNAs. The reconstitution system lays the groundwork for further studies of small RNA-mediated gene silencing in mammals.
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Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.063891.117.
- Received September 4, 2017.
- Accepted September 24, 2017.
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