C. elegans Dicer acts with the RIG-I-like helicase DRH-1 and RDE-4 to cleave dsRNA

Reviewer #1 (Public Review):

Summary:
The authors establish a recombinant insect cell expression and purification scheme for the antiviral Dicer complex of C. elegans. In addition to Dicer-1, the complex harbors two additional proteins, the RIG-I-like helicase DRH-1, and the dsRNA-binding protein RDE-4. The authors show that the complex prefers blunt-end dsRNA over dsRNAs that contain overhangs. Furthermore, whereas ATP-dependent dsRNA cleavage only exacerbates regular dsRNA cleavage activity, the presence of RDE-4 is essential to ATP-dependent and ATP-independent dsRNA cleavage. Single-particle cryo-EM studies of the ternary C. elegans Dicer complex reveal that the N-terminal domain of DRH-1 interacts with the helicase domain of DCR-1, thereby relieving its autoinhibitory state. Lastly, the authors show that the ternary complex is able to processively cleave long dsRNA, an activity primarily relying on the helicase activity of DRH-1.

Strengths:
• First thorough biochemical characterization of the antiviral activity of C. elegans Dicer in complex with the RIG-I-like helicase DRH-1 and the dsRNA-binding protein RDE-4.
• Discovery that RDE-4 is essential to dsRNA processing, whereas ATP hydrolysis is not.
• Discovery of an autoinhibitory role of DRH-1's N-terminal domain (in analogy to the CARD domains of RIG-I).
• First structural insights into the ternary complex DCR-1:DRH-1:RDE-4 by cryo-EM to medium resolution.
• Trap experiments reveal that the ternary DCR-1 complex cleaves blunt-ended dsRNA processively. Likely, the helicase domain of DRH-1 is responsible for this processive cleavage.

Weaknesses:
• Cryo-EM Structure of the ternary Dicer-1:DRH-1:RED-4 complex to only medium resolution.
• High-resolution structure of the C-terminal domain of DRH-1 bound to dsRNA does not reveal the mechanism of how blunt-end dsRNA and overhang-containing one are being discriminated.
• The cryo-EM structure of DCR1:DRH-1:RDE-4 in the presence of ATP only reveals the helicase and CTD domains of DRH-1 bound to dsRNA. No information on dsRNA termini recognition is presented. The paragraph seems detached from the general flow of the manuscript.
• The antiviral DCR-1:DRH-1:RDE-4 complex shows largely homologous activities and regulation than Drosophila Dicer-2.

Reviewer #2 (Public Review):

Summary:
To investigate the evolutionary relationship between the RNAi pathway and innate immunity, this study uses biochemistry and structural biology to investigate the trimeric complex of Dicer-1, DRH-1 (a RIGI homologue), and RDE-4, which exists in C. elegans. The three subunits were co-expressed to promote stable purification of the complex. This complex promoted ATP-dependent cleavage of blunt-ended dsRNAs. A detailed kinetic analysis was also carried out to determine the role of each subunit of the trimeric complex in both the specificity and efficiency of cleavage. These studies indicate that RDE-4 is critical for cleavage while DRC-1 is primarily involved in the specificity of the reaction, and DRH-1 promotes ATP hydrolysis. Finally, a moderate density (6-7 angstrom) cryo-EM structure is presented with attempts to position each of the components.

Strengths:
1. Newly described methods for studying the C. elegans DICER complex.
2. New structure, albeit only moderate resolution.
3. Kinetic study of the complex in the presence and absence of individual subunits and mutations, provides detailed insight into the contribution of each subunit.

Weaknesses:
1. Limited insight due to limited structural resolution.
2. No attempts to extend findings to other Dicer or RLR systems.