Biology subjects

Consalvo, C. D.

Publications and source records attributed to Consalvo, C. D..

3 recordsLinked to original sources

bioRxiv2025

Reconstitution of antiviral Dicer activity in vitro reveals distinct contributions of RDE-4 dsRNA-binding motifs

In C. elegans, antiviral RNA interference (RNAi) relies on the coordinated activity of Dicer (DCR-1), the helicase DRH-1, and the double-stranded RNA (dsRNA)-binding protein, RDE-4, yet the domain-specific contributions of RDE-4 remain unclear. Here, we reconstituted the antiviral complex from independently purified DCR-1*DRH-1 and RDE-4 to define how RDE-4 stabilizes and activates the complex. Addition of recombinant RDE-4 restored ATP hydrolysis and dsRNA cleavage to levels previously observed with the pre-assembled complex, and time-course assays revealed that RDE-4 is essential for maintaining DCR-1*DRH-1 activity. Mutational analysis of RDE-4 revealed that both dsRBM2 and dsRBM3, but not dsRBM1, are required for reconstituting ATP hydrolysis and cleavage. Disruption of the KKxAK motif in dsRBM2 drastically reduced dsRNA affinity and abolished catalytic rescue despite preserving robust binding to DCR-1*DRH-1. Mass photometry and pulldown assays revealed that RDE-4 primarily forms DCR-1 containing complexes, predominantly through interaction with dsRBM3, with no evidence for stable interaction with DRH-1 alone. Functionally, RDE-4 enhanced DRH-1-driven ATP hydrolysis on both 52 and 106 base-pair dsRNAs, but cleavage efficiency showed strong length dependence, implicating dsRNA substrate length as an effector in this system. Our findings establish RDE-4 as an important stabilizer of the antiviral complex and reveal distinct roles for dsRBM2 and dsRBM3 in ATP hydrolysis and dsRNA cleavage. Furthermore, our results suggest that substrate length modulates RDE-4 function, not just alone, but within the antiviral complex. These insights refine our understanding of antiviral RNAi in C. elegans and uncover regulatory mechanisms within the antiviral complex.

biochemistry↗

bioRxiv2025

Biochemical and structural basis of Dicer helicase function unveiled by resurrecting ancient proteins

A fully functional Dicer helicase, present in the modern arthropod, uses energy generated during ATP hydrolysis to power translocation on bound dsRNA, enabling the processive dsRNA cleavage required for efficient antiviral defense. However, modern Dicer orthologs exhibit divergent helicase functions that affect their ability to contribute to antiviral defense, and moreover, mechanisms that couple ATP hydrolysis to Dicer helicase movement on dsRNA remain enigmatic. Here, we used biochemical and structural analyses of ancestrally reconstructed Dicer helicases to map evolution of dsRNA binding affinity, ATP hydrolysis and translocation. We found that loss of affinity for dsRNA occurred early in Dicer evolution, coinciding with a decline in translocation activity, despite preservation of ATP hydrolysis activity, exemplified by the ancient deuterostome Dicer. Ancestral nematode Dicer also exhibited significant decline in ATP hydrolysis and translocation, but studies of antiviral activities in the modern nematode C. elegans indicate Dicer retained a role in antiviral defense by recruiting a second helicase. Cryo-EM analyses of an ancient metazoan Dicer allowed capture of multiple helicase states revealing the mechanism that connects each step of ATP hydrolysis to unidirectional movement along dsRNA. Overall, our study rationalizes the diversity in modern Dicer helicases by connecting ancestral functions to observations in extant enzymes. Significance StatementAmong invertebrates, the contribution of Dicers helicase to recognition and elimination of viral double-stranded RNA varies from phylum to phylum. At the extreme end of the spectrum, vertebrate Dicers show no helicase activity. On the other end, an arthropod ortholog uses helicase translocation to efficiently move double-stranded RNA into Dicers cleavage site. The biochemical and structural basis of Dicers helicase function, as well as the evolutionary events that contribute to a divergence in function, have remained unknown. This study shows how ancient Dicer helicase tightly binds double-stranded RNA and couples ATP hydrolysis to movement along this substrate. In addition, the data reveal how components of this intricate system declined along different clades of animal evolution.

biochemistry↗

bioRxiv2023

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

Abstract/SummaryInvertebrates 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-1s 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.

biochemistry↗