Journal Article

Structural insights into dsRNA processing by Drosophila Dicer-2–Loqs-PD

Ma, Jinbiao, Yuan, Xun, Liu, Nan, Deng, Ting, Wang, Jia, He, Jinqiu, Li, Xiaomin, Huang, Ying, Wang, Hong-Wei, Su, Shichen

2022

Overview

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. Structures of the Dcr-2–Loqs-PD complex while it is processing a double-stranded RNA (dsRNA) substrate elucidate the interactions between Dcr-2 and Loqs-PD, and show that Dcr-2 undergoes substantial conformational changes during a dsRNA-processing cycle.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 101/28 / 13/89 / 631/208/505 / 631/337/505 / 631/45/500 / 631/535/1258/1259 / Adenosine Triphosphate / Animals / ATP / Binding / Binding Sites / Conformation / Cryoelectron Microscopy / DNA helicase / Domains / Double-stranded RNA / Drosophila / Drosophila melanogaster / Drosophila Proteins - chemistry / Drosophila Proteins - metabolism / Drosophila Proteins - ultrastructure / Electron microscopy / Enzymes / Fruit flies / Gene expression / Humanities and Social Sciences / Insects / MicroRNAs / Microscopy / multidisciplinary / Mutation / Phosphates - metabolism / Protein Conformation / Proteins / Ribonuclease III / Ribonuclease III - chemistry / Ribonuclease III - metabolism / Ribonuclease III - ultrastructure / RNA Helicases - chemistry / RNA Helicases - metabolism / RNA Helicases - ultrastructure / RNA, Double-Stranded - chemistry / RNA, Double-Stranded - metabolism / RNA, Double-Stranded - ultrastructure / RNA, Small Interfering - chemistry / RNA, Small Interfering - metabolism / RNA, Small Interfering - ultrastructure / RNA-Binding Proteins - chemistry / RNA-Binding Proteins - metabolism / RNA-Binding Proteins - ultrastructure / RNA-mediated interference / Science / Science (multidisciplinary) / siRNA / Substrates / Translocation