Abstract
In posttranscriptional gene silencing (PTGS), a form of RNA silencing, target RNAs such as mRNAs and viral RNAs are inactivated by RNA-induced silencing complexes (RISCs) through translational repression or endonucleolytic cleavage. A RISC contains an ARGONAUTE family protein (AGO) and a single-stranded small RNA that specifically recognizes the complementary targets. Although the process of RISC assembly influences the induction of PTGS, the molecular mechanisms have not been elucidated. We have developed a plant cell-free RISC assembly system using extracts of evacuolated tobacco BY-2 protoplasts (BYL), and showed that a molecular chaperone HSP90 is required for efficient binding of small RNA duplexes to AGO1. Further analysis characterized a complex containing HSP90, AGO1, and a small RNA duplex, and suggest that, after the HSP90-bound ATP is hydrolyzed, HSP90 is released from the complex containing AGO1 and a small RNA duplex, and the passenger siRNA strand or the miRNA* strand is removed from AGO1. Furthermore, we show evidence suggesting that HSP90 cochaperones are involved in RISC assembly.
