In vitro analyses of the production and activity of secondary small interfering RNAs in C-elegans
In vitro analyses of the production and activity of secondary small interfering RNAs in C-elegans
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DOI：
10.1038/sj.emboj.7601910
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发表时间：
2007-12-12
期刊：
影响因子：
11.4
通讯作者：
Tabara, Hiroaki
中科院分区：
文献类型：
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作者：
Aoki, Kazuma;Moriguchi, Hiromi;Tabara, Hiroaki
在RNA干扰（RNAi）途径中，小干扰RNA（siRNA）作为中间体发挥着重要作用。一级siRNA是由一种称为Dicer的RNaseIII相关酶从触发dsRNA产生的;在一些生物体中，二级siRNA也是通过涉及RNA依赖性RNA聚合酶（RdRP）的过程产生的，其作用于靶mRNA。使用从秀丽隐杆线虫制备的无细胞测定系统，我们分析了次级siRNA的产生和活性。在这种无细胞系统中，RdRP活性作用于mRNA衍生的模板以产生小RNA。RRF-1复合物主要负责RdRP活性，并以不依赖于Dicer的方式合成次级型siRNA分子。值得注意的是，次级型siRNA诱导显著的Slicer活性以比初级型siRNA更有效地切割靶mRNA。Argonaute蛋白CSR-1负责由次级型siRNA诱导的Slicer活性。二级siRNA而不是一级siRNA可能在C.优美的
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.