In vitro reconstitution of the human RISC-loading complex
In vitro reconstitution of the human RISC-loading complex
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DOI：
10.1073/pnas.0710869105
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发表时间：
2008-01-15
影响因子：
11.1
通讯作者：
Doudna, Jennifer A.
中科院分区：
文献类型：
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作者：
MacRae, Ian J.;Ma, Enbo;Doudna, Jennifer A.
通过RNAi靶向基因沉默需要RNA诱导沉默复合物（RISC），其核心组分是与microRNA（miRNA）或siRNA结合的蛋白Argonaute（Ago）。在人类中，Ago2通过称为RISC加载复合物（RLC）的专门组装的作用加载有miRNA，所述RISC加载复合物包括蛋白Ago2、Dicer和TRBP。在这里，我们表明，人类RLC组装自发地在体外从纯化的成分。复合物的形成不需要辅因子或分子伴侣。重组RLC，含有一个拷贝的每种蛋白质，具有切割，切片，引导链选择，和Ago2负载活动观察到的内源性RLC。此外，一旦Ago2装载有miRNA，它倾向于与复合物的其余部分解离。这些结果奠定了基础，为未来的结构和功能解剖RISC加载在人类。
Targeted gene silencing by RNAi requires the RNA-induced silencing complex (RISC), whose core component is the protein Argonaute (Ago) bound to a microRNA (miRNA) or an siRNA. In humans, Ago2 is loaded with miRNAs by the action of a specialized assembly called the RISC-loading complex (RLC), comprising the proteins Ago2, Dicer, and TRBP. Here we show that the human RLC assembles spontaneously in vitro from purified components. No cofactors or chaperones are required for the complex to form. The reconstituted RLC, containing one copy of each protein, has the dicing, slicing, guide-strand selection, and Ago2-loading activities observed for the endogenous RLC. Furthermore, once Ago2 is loaded with an miRNA, it tends to dissociate from the rest of the complex. These results lay the groundwork for future structural and functional dissection of RISC loading in humans.