The RNA-Induced Silencing Complex Is a Mg²⁺-Dependent Endonuclease

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• 作者：Dianne S. Schwarz ; Yukihide Tomari ; Phillip D. Zamore
• 关键词：CELLBIO ; DNA ; RNA
• 刊名：Current Biology
• 出版年：2004
• 出版时间：4 May 2004
• 年：2004
• 卷：14
• 期：9
• 页码：787-791
• 全文大小：226 K

文摘

In the Drosophila and mammalian RNA interference (RNAi) pathways, target RNA destruction is catalyzed by the siRNA-guided, RNA-induced silencing complex (RISC). RISC has been proposed to be an siRNA-directed endonuclease, catalyzing cleavage of a single phosphodiester bond on the RNA target. Although 5′ cleavage products are readily detected for RNAi in vitro, only 3′ cleavage products have been observed in vivo. Proof that RISC acts as an endonuclease requires detection of both 5′ and 3′ cleavage products in a single experimental system. Here, we show that siRNA-programmed RISC generates both 5′ and 3′ cleavage products in vitro; cleavage requires Mg²⁺, but not Ca²⁺, and the cleavage product termini suggest a role for Mg²⁺ in catalysis. Moreover, a single phosphorothioate in place of the scissile phosphate blocks cleavage; the phosphorothioate effect can be rescued by the thiophilic cation Mn²⁺, but not by Ca²⁺ or Mg²⁺. We propose that during catalysis, a Mg²⁺ ion is bound to the RNA substrate through a nonbridging oxygen of the scissile phosphate. The mechanism of endonucleolytic cleavage is not consistent with the mechanisms of the previously identified RISC nuclease, Tudor-SN. Thus, the RISC-component that mediates endonucleolytic cleavage of the target RNA remains to be identified.