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In vitro reconstitution of chaperone-mediated human RISC assembly.

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: Hsp90?, Hsc70, Hop, Dnaja2, and p23. Our data show that ATP hydrolysis by both Hsp90? 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.

SUBMITTER: Naruse K

PROVIDER: S-EPMC5733571 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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In vitro reconstitution of chaperone-mediated human RISC assembly.

Naruse Ken K Matsuura-Suzuki Eriko E Watanabe Mariko M Iwasaki Shintaro S Tomari Yukihide Y

RNA (New York, N.Y.) 20171002 1

To silence target mRNAs, small RNAs and Argonaute (Ago) proteins need to be assembled into RNA-induced silencing complexes (RISCs). Although the assembly of <i>Drosophila melanogaster</i> 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: Hsp90 ...[more]

PMID: 28971854

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