Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA-silencing mechanism in many eukaryotes^1,2. In Drosophila, an RNase III enzyme Dicer-2 (Dcr-2), aided by its cofactor Loquacious-PD (Loqs-PD), has an important role in generating 21 bp siRNA duplexes from long double-stranded RNAs (dsRNAs)^3,4. ATP hydrolysis by the helicase domain of Dcr-2 is critical to the successful processing of a long dsRNA into consecutive siRNA duplexes^5,6. Here we report the cryo-electron microscopy structures of Dcr-2–Loqs-PD in the apo state and in multiple states in which it is processing a 50 bp dsRNA substrate. The structures elucidated interactions between Dcr-2 and Loqs-PD, and substantial conformational changes of Dcr-2 during a dsRNA-processing cycle. The N-terminal helicase and domain of unknown function 283 (DUF283) domains undergo conformational changes after initial dsRNA binding, forming an ATP-binding pocket and a 5′-phosphate-binding pocket. The overall conformation of Dcr-2–Loqs-PD is relatively rigid during translocating along the dsRNA in the presence of ATP, whereas the interactions between the DUF283 and RIIIDb domains prevent non-specific cleavage during translocation by blocking the access of dsRNA to the RNase active centre. Additional ATP-dependent conformational changes are required to form an active dicing state and precisely cleave the dsRNA into a 21 bp siRNA duplex as confirmed by the structure in the post-dicing state. Collectively, this study revealed the molecular mechanism for the full cycle of ATP-dependent dsRNA processing by Dcr-2–Loqs-PD.

小干扰 RNA (siRNA) 是 RNA 干扰 (RNAi) 的关键组成部分，RNA 干扰是许多真核生物中的一种保守 RNA 沉默机制^1,2。在果蝇中，核糖核酸酶 III 酶 Dicer-2 (Dcr-2) 在其辅助因子 Loquacious-PD (Loqs-PD) 的帮助下，在从长双链 RNA (dsRNA) 3 生成 21 bp siRNA 双链体方面具有重要作用^{， 4} . Dcr-2 的解旋酶结构域对 ATP 的水解对于将长 dsRNA 成功加工成连续的 siRNA 双链体至关重要^5,6. 在这里，我们报告了 Dcr-2–Loqs-PD 在 apo 状态和处理 50 bp dsRNA 底物的多种状态下的冷冻电子显微镜结构。这些结构阐明了 Dcr-2 和 Loqs-PD 之间的相互作用，以及 Dcr-2 在 dsRNA 加工周期中的显着构象变化。N 末端解旋酶和未知功能域 283 (DUF283) 结构域在初始 dsRNA 结合后发生构象变化，形成 ATP 结合口袋和 5'-磷酸盐结合口袋。Dcr-2–Loqs-PD 的整体构象在存在 ATP 的情况下沿 dsRNA 转运期间相对刚性，而 DUF283 和 RIIIDb 结构域之间的相互作用通过阻止 dsRNA 进入 RNase 来防止转运期间的非特异性切割活性中心。需要额外的 ATP 依赖性构象变化才能形成活跃的切割状态，并精确地将 dsRNA 切割成 21 bp 的 siRNA 双链体，正如切割后状态的结构所证实的那样。总的来说，这项研究揭示了 Dcr-2–Loqs-PD 对 ATP 依赖性 dsRNA 加工全周期的分子机制。