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

被引:165
作者
Aoki, Kazuma [2 ]
Moriguchi, Hiromi [2 ]
Yoshioka, Tomoko [2 ]
Okawa, Katsuya [2 ]
Tabara, Hiroaki [1 ,2 ]
机构
[1] Kyoto Univ, Grad Sch Med, Kyoto 6068501, Japan
[2] Kyoto Univ, Grad Sch Med, HMRO, Kyoto, Japan
关键词
Argonaute; RdRP; RNAi; siRNA;
D O I
10.1038/sj.emboj.7601910
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:5007 / 5019
页数:13
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