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 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.