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Article

  • The EMBO Journal (2007) 26, 5007 - 5019
  • doi:10.1038/sj.emboj.7601910

Published online: 15 November 2007

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

Kazuma Aoki1, Hiromi Moriguchi1, Tomoko Yoshioka1, Katsuya Okawa1 and Hiroaki Tabara1,2

  1. Graduate School of Medicine, HMRO, Kyoto University, Kyoto, Japan
  2. PRESTO, Japan Science and Technology Agency, Saitama, Japan

Correspondence to:

Hiroaki Tabara, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Sakyo, Building E, Kyoto 606-8501, Japan. Tel.: +81 75 753 9305; Fax: +81 75 753 9311; E-mail: htabara@hmro.med.kyoto-u.ac.jp

Received 29 May 2007; Accepted 10 October 2007

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.

  • Keywords:

    • Argonaute,
    • RdRP,
    • RNAi,
    • siRNA