Cyclophilin 40 facilitates HSP90-mediated RISC assembly in plants

Posttranscriptional gene silencing is mediated by RNA‐induced silencing complexes (RISCs) that contain AGO proteins and single‐stranded small RNAs. The assembly of plant AGO1‐containing RISCs depends on the molecular chaperone HSP90. Here, we demonstrate that cyclophilin 40 (CYP40), protein phosphatase 5 (PP5), and several other proteins with the tetratricopeptide repeat (TPR) domain associates with AGO1 in an HSP90‐dependent manner in extracts of evacuolated tobacco protoplasts (BYL). Intriguingly, CYP40, but not the other TPR proteins, could form a complex with small RNA duplex‐bound AGO1. Moreover, CYP40 that was synthesized by in‐vitro translation using BYL uniquely facilitated binding of small RNA duplexes to AGO1, and as a result, increased the amount of mature RISCs that could cleave target RNAs. CYP40 was not contained in mature RISCs, indicating that the association is transient. Addition of PP5 or cyclophilin‐binding drug cyclosporine A prevented the association of endogenous CYP40 with HSP90–AGO1 complex and inhibited RISC assembly. These results suggest that a complex of AGO1, HSP90, CYP40, and a small RNA duplex is a key intermediate of RISC assembly in plants. AGO1 plays an important role in the miRNA pathway in plants; however, relatively little is known about the assembly of the AGO1–miRISC complex in this organism. This study shows that cyclophilin 40 promotes the association of HSP90–AGO1 with duplexed small RNAs, while PP5 opposes this effect.