State Key Laboratory of Genetic Engineering, Collaborative Innovation Centre of Genetics and Development, Department of Biochemistry and Biophysics, Institute of Plant Biology, School of Life Sciences, Fudan University, 200438, Shanghai, China.
Shanghai Key Laboratory of Biliary Tract Disease Research, Shanghai Research Center of Biliary Tract Disease, Department of General Surgery, Xinhua Hospital, Affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Nat Commun. 2023 Aug 26;14(1):5228. doi: 10.1038/s41467-023-40919-1.
Small interference RNAs are the key components of RNA interference, a conserved RNA silencing or viral defense mechanism in many eukaryotes. In Drosophila melanogaster, Dicer-2 (DmDcr-2)-mediated RNAi pathway plays important roles in defending against viral infections and protecting genome integrity. During the maturation of siRNAs, two cofactors can regulate DmDcr-2's functions: Loqs-PD that is required for dsRNA processing, and R2D2 that is essential for the subsequent loading of siRNAs into effector Ago2 to form RISC complexes. However, due to the lack of structural information, it is still unclear whether R2D2 and Loqs-PD affect the functions of DmDcr-2 simultaneously. Here we present several cryo-EM structures of DmDcr-2/R2D2/Loqs-PD complex bound to dsRNAs with various lengths by the Helicase domain. These structures revealed that R2D2 and Loqs-PD can bind to different regions of DmDcr-2 without interfering with each other. Furthermore, the cryo-EM results demonstrate that these complexes can form large oligomers and assemble into fibers. The formation and depolymerization of these oligomers are associated with ATP hydrolysis. These findings provide insights into the structural mechanism of DmDcr-2 and its cofactors during siRNA processing.
小干扰 RNA 是 RNA 干扰的关键组成部分，RNA 干扰是许多真核生物中保守的 RNA 沉默或病毒防御机制。在黑腹果蝇中，Dicer-2（DmDcr-2）介导的 RNAi 途径在抵御病毒感染和保护基因组完整性方面发挥着重要作用。在 siRNA 的成熟过程中，有两种辅助因子可以调节 DmDcr-2 的功能：Loqs-PD，其对于 dsRNA 加工是必需的；R2D2，其对于随后将 siRNA 加载到效应物 Ago2 中形成 RISC 复合物是必不可少的。然而，由于缺乏结构信息，目前尚不清楚 R2D2 和 Loqs-PD 是否同时影响 DmDcr-2 的功能。在这里，我们通过解旋酶结构域展示了几种与不同长度 dsRNA 结合的 DmDcr-2/R2D2/Loqs-PD 复合物的冷冻电镜结构。这些结构揭示了 R2D2 和 Loqs-PD 可以结合到 DmDcr-2 的不同区域而不会相互干扰。此外，冷冻电镜结果表明这些复合物可以形成大的寡聚体并组装成纤维。这些寡聚体的形成和解聚与 ATP 水解有关。这些发现为 siRNA 加工过程中 DmDcr-2 及其辅助因子的结构机制提供了新的见解。
