title: Cyclophilin 40 facilitates HSP90-mediated RISC assembly in plants.
abstract: 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.
authors: Division of Plant Sciences, Plant-Microbe Interactions Research Unit, National Institute of Agrobiological Sciences (NIAS), Ibaraki, Japan.
authors: Taichiro
authors: T
authors: Iki
authors: Manabu
authors: M
authors: Yoshikawa
authors: Tetsuo
authors: T
authors: Meshi
authors: Masayuki
authors: M
authors: Ishikawa
mesh_terms: D060565:Argonaute Proteins / Q000378:metabolism*; D000081406:Peptidyl-Prolyl Isomerase F; D021983:Cyclophilins / Q000502:physiology*; D016572:Cyclosporine / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000502:physiology*; D035683:MicroRNAs / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010940:Plant Proteins / Q000502:physiology*; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D034622:RNA Interference / Q000502:physiology*; D018749:RNA, Plant / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000187:drug effects / Q000378:metabolism*; D022021:Tacrolimus Binding Proteins / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
chemical_list: D060565:Argonaute Proteins; D000081406:Peptidyl-Prolyl Isomerase F; D018841:HSP90 Heat-Shock Proteins; D035683:MicroRNAs; D009687:Nuclear Proteins; D010940:Plant Proteins; D018749:RNA, Plant; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D016572:Cyclosporine; D010749:Phosphoprotein Phosphatases; C089641:protein phosphatase 5; D021983:Cyclophilins; D022021:Tacrolimus Binding Proteins