In vitro Assembly of Plant RNA-induced Silencing Complexes

Abstract

RNA-induced silencing complexes (RISCs) play central roles in posttranscriptional gene silencing. RISCs invariably contain ARGONAUTE (AGO) family proteins and single-stranded small RNAs. In plants, the mechanism of RISC assembly has remained elusive due to the lack of cell-free systems that recapitulate the in vivo process. We demonstrate that a cleavage-competent RISC can be assembled with a synthetic small interfering RNA (siRNA) duplex in the extracts of evacuolated tobacco BY-2 protoplasts (BYL). In the BYL system, the siRNA duplex was incorporated into AGO1 by a mechanism requiring ATP, Mg²⁺ and the molecular chaperone HSP90, and the removal of passenger strand of siRNA duplex depended on the RNase activity of AGO1. Directed by the guide strand siRNA sequestered to AGO1, the assembled RISC bound and cleaved the complementary target RNA.