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

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Small interfering RNAs (siRNAs) mediate RNA interference (RNAi), a crucial gene-silencing pathway in eukaryotes.
• Drosophila Dicer-2 (Dcr-2), with its cofactor Loquacious-PD (Loqs-PD), generates siRNAs from long double-stranded RNAs (dsRNAs).
• ATP hydrolysis by Dcr-2's helicase domain is essential for efficient dsRNA processing into siRNA duplexes.

Purpose of the Study:

• To elucidate the structural mechanisms underlying ATP-dependent dsRNA processing by the Dcr-2-Loqs-PD complex.
• To visualize the conformational dynamics of Dcr-2 during the siRNA generation cycle.
• To understand how Dcr-2-Loqs-PD precisely cleaves dsRNA into 21 bp siRNA duplexes.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was used to determine the structures of Dcr-2-Loqs-PD.
• Structures were obtained for the apo state and multiple substrate-bound processing states, including post-dicing.
• The study analyzed conformational changes and molecular interactions during dsRNA translocation and cleavage.

Main Results:

• Structures revealed interactions between Dcr-2 and Loqs-PD and significant Dcr-2 conformational shifts during dsRNA processing.
• ATP binding induces changes in Dcr-2's helicase and DUF283 domains, forming key binding pockets.
• Dcr-2-Loqs-PD exhibits relative rigidity during ATP-dependent translocation, with DUF283-RIIIDb interactions preventing premature cleavage.

Conclusions:

• The study provides a detailed molecular mechanism for the ATP-dependent dsRNA processing cycle by Dcr-2-Loqs-PD.
• Structural insights explain how Dcr-2 precisely generates 21 bp siRNA duplexes.
• This work clarifies the roles of ATP hydrolysis and protein-protein interactions in RNAi pathway execution.