In vitro studies provide insight into effects of Dicer-2 helicase mutations in Drosophila melanogaster.

In vitro, Drosophila melanogaster Dicer-2 (Dcr-2) uses its helicase domain to initiate processing of dsRNA with blunt (BLT) termini, and its Platform•PAZ domain to initiate processing of dsRNA with 3' overhangs (ovrs). To understand the relationship of these in vitro observations to roles of Dcr-2 in vivo, we compared in vitro effects of two helicase mutations to their impact on production of endogenous and viral siRNAs in flies. Consistent with the importance of the helicase domain in processing BLT dsRNA, both point mutations eliminated processing of BLT, but not 3'ovr, dsRNA in vitro. However, the mutations had different effects in vivo. A point mutation in the Walker A motif of the Hel1 subdomain, G31R, largely eliminated production of siRNAs in vivo, while F225G, located in the Hel2 subdomain, showed reduced levels of endogenous siRNAs, but did not significantly affect virus-derived siRNAs. In vitro assays monitoring dsRNA cleavage, dsRNA binding, ATP hydrolysis, and binding of the accessory factor Loquacious-PD provided insight into the different effects of the mutations on processing of different sources of dsRNA in flies. Our in vitro studies suggest effects of the mutations in vivo relate to their effects on ATPase activity, dsRNA binding, and interactions with Loquacious-PD. Our studies emphasize the importance of future studies to characterize dsRNA termini as they exist in Drosophila and other animals.

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

Invertebrates use the endoribonuclease Dicer to cleave viral dsRNA during antiviral defense, while vertebrates use RIG-I-like Receptors (RLRs), which bind viral dsRNA to trigger an interferon response. While some invertebrate Dicers act alone during antiviral defense, C. elegans Dicer acts in a complex with a dsRNA binding protein called RDE-4, and an RLR ortholog called DRH-1. We used biochemical and structural techniques to provide mechanistic insight into how these proteins function together. We found RDE-4 is important for ATP-independent and ATP-dependent cleavage reactions, while helicase domains of both DCR-1 and DRH-1 contribute to ATP-dependent cleavage. DRH-1 plays the dominant role in ATP hydrolysis, and like mammalian RLRs, has an N-terminal domain that functions in autoinhibition. A cryo-EM structure indicates DRH-1 interacts with DCR-1's helicase domain, suggesting this interaction relieves autoinhibition. Our study unravels the mechanistic basis of the collaboration between two helicases from typically distinct innate immune defense pathways.

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

Invertebrates use the endoribonuclease Dicer to cleave viral dsRNA during antiviral defense, while vertebrates use RIG-I-like Receptors (RLRs), which bind viral dsRNA to trigger an interferon response. While some invertebrate Dicers act alone during antiviral defense, Caenorhabditis elegans Dicer acts in a complex with a dsRNA binding protein called RDE-4, and an RLR ortholog called DRH-1. We used biochemical and structural techniques to provide mechanistic insight into how these proteins function together. We found RDE-4 is important for ATP-independent and ATP-dependent cleavage reactions, while helicase domains of both DCR-1 and DRH-1 contribute to ATP-dependent cleavage. DRH-1 plays the dominant role in ATP hydrolysis, and like mammalian RLRs, has an N-terminal domain that functions in autoinhibition. A cryo-EM structure indicates DRH-1 interacts with DCR-1's helicase domain, suggesting this interaction relieves autoinhibition. Our study unravels the mechanistic basis of the collaboration between two helicases from typically distinct innate immune defense pathways.

Lysate-to-grid: Rapid Isolation of Native Complexes from Budding Yeast for Cryo-EM Imaging.

Single-particle electron cryo-microscopy (cryo-EM) is an effective tool to determine high-resolution structures of macromolecular complexes. Its lower requirements for sample concentration and purity make it an accessible method to determine structures of low-abundant protein complexes, such as those isolated from native sources. While there are many approaches to protein purification for cryo-EM, attaining suitable particle quality and abundance is generally the major bottleneck to the typical single-particle project workflow. Here, we present a protocol using budding yeast ( S. cerevisiae ), in which a tractable immunoprecipitation tag (3xFLAG) is appended at the endogenous locus of a gene of interest (GOI). The modified gene is expressed under its endogenous promoter, and cells are grown and harvested using standard procedures. Our protocol describes the steps in which the tagged proteins and their associated complexes are isolated within three hours of thawing cell lysates, after which the recovered proteins are used directly for cryo-EM specimen preparation. The prioritization of speed maximizes the ability to recover intact, scarce complexes. The protocol is generalizable to soluble yeast proteins that tolerate C-terminal epitope tags. Graphical abstract Overview of lysate-to-grid workflow. Yeast cells are transformed to express a tractable tag on a gene of interest. Following cell culture and lysis, particles of interest are rapidly isolated by co-immunoprecipitation and prepared for cryo-EM imaging (created with BioRender.com).

Loquacious-PD regulates the terminus-dependent molecular recognition of Dicer-2 toward double-stranded RNA.

Dicer-2 cleaves double-stranded RNA into siRNAs in a terminus-dependent manner as part of D. melanogaster's RNA interference pathway. Using ultrafast fluorescence, we probe the local environment of chromophores at the dsRNA terminus upon binding by Dicer-2 and interrogate the effects of Loquacious-PD, an accessory protein. We find substrate-selective modes of molecular recognition that distinguish between blunt and 3'overhang termini, but whose differences are greatly reduced by Loquacious-PD. These results connect the molecular recognition properties of Dicer-2 to its selective processing of dsRNAs with different termini and to its need for Loquacious-PD to efficiently produce endogenous siRNAs.

Dicer's Helicase Domain: A Meeting Place for Regulatory Proteins.

The function of Dicer's helicase domain has been enigmatic since its discovery. Why do only some Dicers require ATP, despite a high degree of sequence conservation in their helicase domains? We discuss evolutionary considerations based on differences between vertebrate and invertebrate antiviral defense, and how the helicase domain has been co-opted in extant organisms as the binding site for accessory proteins. Many accessory proteins are double-stranded RNA binding proteins, and we propose models for how they modulate Dicer function and catalysis.