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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Animals, Genetically Modified
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Caenorhabditis elegans / genetics*
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Caenorhabditis elegans Proteins / genetics
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Caenorhabditis elegans Proteins / metabolism
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Cell-Free System
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DEAD-box RNA Helicases / genetics
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DEAD-box RNA Helicases / metabolism
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Endoribonucleases / genetics
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Endoribonucleases / metabolism
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Eukaryotic Initiation Factor-2 / genetics
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Eukaryotic Initiation Factor-2 / metabolism
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Gene Expression Regulation
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Multiprotein Complexes / metabolism
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RNA Interference*
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RNA, Helminth / genetics
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RNA, Helminth / metabolism*
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RNA, Messenger / metabolism
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RNA, Small Interfering / genetics
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RNA, Small Interfering / metabolism*
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / metabolism
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RNA-Dependent RNA Polymerase / genetics
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RNA-Dependent RNA Polymerase / metabolism
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Ribonuclease III
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Transgenes
Substances
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Caenorhabditis elegans Proteins
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Eukaryotic Initiation Factor-2
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Multiprotein Complexes
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RDE-4 protein, C elegans
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RNA, Helminth
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RNA, Messenger
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RNA, Small Interfering
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RNA-Binding Proteins
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Recombinant Fusion Proteins
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Drh-3 protein, C elegans
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RNA-Dependent RNA Polymerase
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Endoribonucleases
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dcr-1 protein, C elegans
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Ribonuclease III
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DEAD-box RNA Helicases