title: In vitro analyses of the production and activity of secondary small interfering RNAs in C. elegans.
abstract: In the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) play important roles as intermediates. Primary siRNAs are produced from trigger dsRNAs by an RNaseIII-related enzyme called Dicer; in some organisms, secondary siRNAs are also produced by processes involving RNA-dependent RNA polymerases (RdRPs), which act on target mRNAs. Using a cell-free assay system prepared from Caenorhabditis elegans, we analyzed the production and activity of secondary siRNAs. In this cell-free system, RdRP activity acts on mRNA-derived templates to produce small RNAs. The RRF-1 complex is predominantly responsible for the RdRP activity, and synthesizes secondary-type siRNA molecules in a Dicer-independent manner. Notably, secondary-type siRNAs induce a prominent Slicer activity to cleave target mRNAs far more effectively than primary-type siRNAs. An Argonaute protein, CSR-1, is responsible for the Slicer activity induced by secondary-type siRNAs. Secondary rather than primary siRNAs may play a major role in the destabilization of target transcripts during RNAi in C. elegans.
authors.affiliation: Graduate School of Medicine, HMRO, Kyoto University, Kyoto, Japan.
authors.forename: Kazuma
authors.initials: K
authors.lastname: Aoki
authors.forename: Hiromi
authors.initials: H
authors.lastname: Moriguchi
authors.forename: Tomoko
authors.initials: T
authors.lastname: Yoshioka
authors.forename: Katsuya
authors.initials: K
authors.lastname: Okawa
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tabara
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D046912:Multiprotein Complexes / Q000378:metabolism; D034622:RNA Interference; D018510:RNA, Helminth / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III; D019076:Transgenes
chemical_list: D029742:Caenorhabditis elegans Proteins; D015852:Eukaryotic Initiation Factor-2; D046912:Multiprotein Complexes; C461645:RDE-4 protein, C elegans; D018510:RNA, Helminth; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C524398:Drh-3 protein, C elegans; D012324:RNA-Dependent RNA Polymerase; D004722:Endoribonucleases; C118116:dcr-1 protein, C elegans; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases