title: Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes.
abstract: In the Drosophila and mammalian RNA interference pathways, siRNAs direct the protein Argonaute2 (Ago2) to cleave corresponding mRNA targets, silencing their expression. Ago2 is the catalytic component of the RNAi enzyme complex, RISC. For each siRNA duplex, only one strand, the guide, is assembled into the active RISC; the other strand, the passenger, is destroyed. An ATP-dependent helicase has been proposed first to separate the two siRNA strands, then the resulting single-stranded guide is thought to bind Ago2. Here, we show that Ago2 instead directly receives the double-stranded siRNA from the RISC assembly machinery. Ago2 then cleaves the siRNA passenger strand, thereby liberating the single-stranded guide. For siRNAs, virtually all RISC is assembled through this cleavage-assisted mechanism. In contrast, passenger-strand cleavage is not important for the incorporation of miRNAs that derive from mismatched duplexes.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Matranga
authors.forename: Yukihide
authors.initials: Y
authors.lastname: Tomari
authors.forename: Chanseok
authors.initials: C
authors.lastname: Shin
authors.forename: David P
authors.initials: DP
authors.lastname: Bartel
authors.forename: Phillip D
authors.initials: PD
authors.lastname: Zamore
mesh_terms: D000818:Animals; D060565:Argonaute Proteins; D001483:Base Sequence; D002384:Catalysis; D002460:Cell Line; D002474:Cell-Free System; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005260:Female; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D009154:Mutation / Q000235:genetics; D009419:Nerve Tissue Proteins; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D010053:Ovary / Q000378:metabolism; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000235:genetics / Q000378:metabolism*; D043244:Ribonuclease III; D013873:Thionucleotides / Q000737:chemistry
chemical_list: C433807:AGO2 protein, Drosophila; D060565:Argonaute Proteins; D029721:Drosophila Proteins; C429034:FSD1 protein, human; D035683:MicroRNAs; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D009841:Oligonucleotides; C478068:R2D2 protein, Drosophila; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D034743:RNA-Induced Silencing Complex; D013873:Thionucleotides; D008345:Manganese; C478069:DCR-2 protein, Drosophila; D043244:Ribonuclease III; D020365:RNA Helicases; D008274:Magnesium