In vitro analyses of the production and activity of secondary small interfering RNAs in C. elegans.
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.
Aoki
Kazuma
K
Graduate School of Medicine, HMRO, Kyoto University, Kyoto, Japan.
Moriguchi
Hiromi
H
Yoshioka
Tomoko
T
Okawa
Katsuya
K
Tabara
Hiroaki
H
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
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