西亚试剂：Defining fundamental steps in the assembly of theDrosophila

Small RNAs such as small interfering RNAs (siRNAs) and microRNAs (miRNAs) silence the expression of their complementary target messenger RNAs via the formation of effector RNA-induced silencing complexes (RISCs), which contain Argonaute (Ago) family proteins at their core. Although loading of siRNA duplexes into Drosophila Ago2 requires the Dicer-2–R2D2 heterodimer and the Hsc70/Hsp90 (Hsp90 also known as Hsp83) chaperone machinery, the details of RISC assembly remain unclear. Here we reconstitute RISC assembly using only Ago2, Dicer-2, R2D2, Hsc70, Hsp90, Hop, Droj2 (an Hsp40 homologue) and p23. By following the assembly of single RISC molecules, we find that, in the absence of the chaperone machinery, an siRNA bound to Dicer-2–R2D2 associates with Ago2 only transiently. The chaperone machinery extends the dwell time of the Dicer-2–R2D2–siRNA complex on Ago2, in a manner dependent on recognition of the 5′-phosphate on the siRNA guide strand. We propose that the chaperone machinery supports a productive state of Ago2, allowing it to load siRNA duplexes from Dicer-2–R2D2 and thereby assemble RISC.

 RNA沉默中涉及的中心效应子是RISC，它是由RNA诱导的沉默复合物，由结合到一个“小干涉RNA”(siRNA)上的一种Argonaute (Ago)蛋白组成。Yukihide Tomari及同事现在利用8种纯化的蛋白重构了单一果蝇RISC分子的聚合体。他们发现，伴侣分子有助于稳定siRNA(它最初与Dicer-2和R2D2的一个复合物结合在一起)与Ago2之间的相互作用。这种稳定化使Ago2能够识别siRNA引导链的5′-磷酸盐，在此之后该引导链被转移到Ago2上。

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