ABSTRACT
It was first shown that DNA damage induction in mitomycin C-treated HeLa cells leads to a change in the selection of 5p and 3p microRNA duplex strands in the formation of the RNA-induced silencing complex (RISC).
Subject(s)
DNA Damage/genetics , DNA Damage/physiology , MicroRNAs/genetics , MicroRNAs/metabolism , DNA Damage/drug effects , HeLa Cells , Humans , Mitomycin/toxicity , Nucleic Acid Synthesis Inhibitors/toxicity , RNA-Induced Silencing Complex/drug effects , RNA-Induced Silencing Complex/genetics , RNA-Induced Silencing Complex/metabolismABSTRACT
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.
