title: Defining fundamental steps in the assembly of the Drosophila RNAi enzyme complex.
abstract: Small RNAs such as small interfering RNAs (siRNAs) and microRNAs (miRNAs) silence the expression of their complementary target messenger RNAs via the formation of effector RNA-induced silencing complexes (RISCs), which contain Argonaute (Ago) family proteins at their core. Although loading of siRNA duplexes into Drosophila Ago2 requires the Dicer-2-R2D2 heterodimer and the Hsc70/Hsp90 (Hsp90 also known as Hsp83) chaperone machinery, the details of RISC assembly remain unclear. Here we reconstitute RISC assembly using only Ago2, Dicer-2, R2D2, Hsc70, Hsp90, Hop, Droj2 (an Hsp40 homologue) and p23. By following the assembly of single RISC molecules, we find that, in the absence of the chaperone machinery, an siRNA bound to Dicer-2-R2D2 associates with Ago2 only transiently. The chaperone machinery extends the dwell time of the Dicer-2-R2D2-siRNA complex on Ago2, in a manner dependent on recognition of the 5'-phosphate on the siRNA guide strand. We propose that the chaperone machinery supports a productive state of Ago2, allowing it to load siRNA duplexes from Dicer-2-R2D2 and thereby assemble RISC.
authors.affiliation: Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan.
authors.forename: Shintaro
authors.initials: S
authors.lastname: Iwasaki
authors.affiliation: Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan.
authors.forename: Hiroshi M
authors.initials: HM
authors.lastname: Sasaki
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Yuriko
authors.initials: Y
authors.lastname: Sakaguchi
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Tadakuma
authors.affiliation: 1] Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan. [2] Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan.
authors.forename: Yukihide
authors.initials: Y
authors.lastname: Tomari
mesh_terms: D000818:Animals; D060565:Argonaute Proteins / Q000378:metabolism; D029721:Drosophila Proteins / Q000378:metabolism; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics*; D006360:Heat-Shock Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D053612:Janus Kinases / Q000378:metabolism; D011485:Protein Binding; D020365:RNA Helicases / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000737:chemistry* / Q000378:metabolism*; D043244:Ribonuclease III / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D060565:Argonaute Proteins; D029721:Drosophila Proteins; D006360:Heat-Shock Proteins; D053612:Janus Kinases; D020365:RNA Helicases; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D034743:RNA-Induced Silencing Complex; D043244:Ribonuclease III; D014157:Transcription Factors; D000094704:RNA, Guide, CRISPR-Cas Systems; C433807:AGO2 protein, Drosophila; C478068:R2D2 protein, Drosophila; C085845:hop protein, Drosophila; C478069:DCR-2 protein, Drosophila
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