Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Nat Struct Mol Biol. 2012 Jan 10;19(2):145-51. doi: 10.1038/nsmb.2232.
Small RNAs, such as microRNAs and small interfering RNAs, act through Argonaute (Ago) proteins as a part of RNA-induced silencing complexes (RISCs). To make RISCs, Ago proteins bind and subsequently unwind small RNA duplexes, finally leaving one strand stably incorporated. Here we identified the N domain of human AGO2 as the initiator of duplex unwinding during RISC assembly. We discovered that a functional N domain is strictly required for small RNA duplex unwinding but not for precedent duplex loading or subsequent target cleavage. We postulate that RISC assembly is tripartite, comprising (i) RISC loading, whereby Ago undergoes conformational opening and loads a small RNA duplex, forming pre-RISC; (ii) wedging, whereby the end of the duplex is pried open through active wedging by the N domain, in preparation for unwinding; and (iii) unwinding, whereby the passenger strand is removed through slicer-dependent or slicer-independent unwinding, forming mature RISC.
小 RNA （如 microRNA 和 small interfering RNA）通过 Argonaute （AGO）蛋白作为 RNA 诱导沉默复合物（RISCs）的一部分发挥作用。为了形成 RISCs，AGO 蛋白结合并随后解双链小 RNA ，最终使一条链稳定地结合。在这里，我们鉴定出人 AGO2 的 N 结构域是 RISC 组装过程中双链解旋的起始子。我们发现，功能性 N 结构域严格需要用于小 RNA 双链解旋，但不需要用于先例双链加载或随后的靶标切割。我们推测 RISC 组装是三分体的，包括（i）RISC 加载，其中 Ago 经历构象开放并加载小 RNA 双链，形成 pre-RISC；（ii）楔入，其中双链的末端通过 N 结构域的主动楔入打开，为解旋做准备；（iii）解旋，其中通过依赖于 slicer 的或不依赖于 slicer 的解旋去除过客链，形成成熟的 RISC。
