Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Nat Struct Mol Biol. 2010 Jan;17(1):17-23. doi: 10.1038/nsmb.1733. Epub 2009 Dec 6.
The assembly of RNA-induced silencing complex (RISC) is a key process in small RNA-mediated gene silencing. In humans, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are incorporated into RISCs containing the Argonaute (AGO) subfamily proteins Ago1-4. Previous studies have proposed that, unlike Drosophila melanogaster RISC assembly pathways, human RISC assembly is coupled with dicing and is independent of ATP. Here we show by careful reexamination that, in humans, RISC assembly and dicing are uncoupled, and ATP greatly facilitates RISC loading of small-RNA duplexes. Moreover, all four human AGO proteins show remarkably similar structural preferences for small-RNA duplexes: central mismatches promote RISC loading, and seed or 3'-mid (guide position 12-15) mismatches facilitate unwinding. All these features of human AGO proteins are highly reminiscent of fly Ago1 but not fly Ago2.
RNA 诱导沉默复合物（RISC）的组装是小 RNA 介导的基因沉默的关键过程。在人类中，小干扰 RNA（siRNA）和 microRNA（miRNA）被整合到含有 Argonaute（AGO）亚家族蛋白 Ago1-4 的 RISC 中。先前的研究表明，与果蝇 RISC 组装途径不同，人类 RISC 组装与 Dicer 的切割过程解耦，并且不依赖于 ATP。在这里，我们通过仔细的重新检查表明，在人类中，RISC 组装和 Dicer 的切割过程是解耦的，并且 ATP 极大地促进了小 RNA 双链体加载到 RISC 中。此外，所有四种人类 AGO 蛋白对小 RNA 双链体表现出非常相似的结构偏好：中心错配促进 RISC 加载，而种子或 3'-mid（引导位置 12-15）错配促进解链。人类 AGO 蛋白的所有这些特征都非常类似于果蝇 Ago1，但不同于果蝇 Ago2。
