In vitro analyses of the production and activity of secondary small interfering RNAs in C. elegans

EMBO J. 2007 Dec 12;26(24):5007-19. doi: 10.1038/sj.emboj.7601910. Epub 2007 Nov 15.


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

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Caenorhabditis elegans / genetics*
  • Caenorhabditis elegans Proteins / genetics
  • Caenorhabditis elegans Proteins / metabolism
  • Cell-Free System
  • DEAD-box RNA Helicases / genetics
  • DEAD-box RNA Helicases / metabolism
  • Endoribonucleases / genetics
  • Endoribonucleases / metabolism
  • Eukaryotic Initiation Factor-2 / genetics
  • Eukaryotic Initiation Factor-2 / metabolism
  • Gene Expression Regulation
  • Multiprotein Complexes / metabolism
  • RNA Interference*
  • RNA, Helminth / genetics
  • RNA, Helminth / metabolism*
  • RNA, Messenger / metabolism
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • RNA-Dependent RNA Polymerase / genetics
  • RNA-Dependent RNA Polymerase / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Ribonuclease III
  • Transgenes


  • Caenorhabditis elegans Proteins
  • Eukaryotic Initiation Factor-2
  • Multiprotein Complexes
  • RDE-4 protein, C elegans
  • RNA, Helminth
  • RNA, Messenger
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • Recombinant Fusion Proteins
  • Drh-3 protein, C elegans
  • RNA-Dependent RNA Polymerase
  • Endoribonucleases
  • dcr-1 protein, C elegans
  • Ribonuclease III
  • DEAD-box RNA Helicases