In vitro reconstitution of chaperone-mediated human RISC assembly
- ↵* Corresponding author; email: tomari{at}iam.u-tokyo.ac.jp
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 5 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 5 recombinant chaperone proteins: Hsp90β, Hsc70, Hop, Dnaja2, and p23. This reconstitution system reflected the dependence on ATP hydrolysis, a hallmark of RISC assembly. Whereas the Dicer-2/R2D2 heterodimer is a prerequisite for fly Ago2-RISC assembly, human Ago2-RISC can be formed in the absence of Dicer or TRBP in our reconstituted system. Our method provides a versatile framework for further studies of small RNA-mediated gene silencing in mammals.
- Received September 4, 2017.
- Accepted September 24, 2017.
- Published by Cold Spring Harbor Laboratory Press for the RNA Society
This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
