Biology subjects

Aruscavage, P. J.

Publications and source records attributed to Aruscavage, P. J..

5 recordsLinked to original sources

bioRxiv2026

C. elegans Dicer stacks with the RIG-I-like receptor DRH-1 to cleave dsRNA

In prior studies we showed that the nematode ancestor of Dicer's helicase domain had minimal ATP hydrolysis, translocation and dsRNA binding activity, and in extant C. elegans the RIG-I like receptor (RLR) DRH-1, and the dsRNA binding protein RDE-4, were co-opted to provide these activities. Here we report cleavage-competent cryo-EM structures of the antiviral complex (AVC; DCR-1{middle dot}DRH-1{middle dot}RDE-4), in the absence (3.2[A]) and presence (3.0[A] of ATP. A key feature of both structures is stacking of DCR-1's helicase with the helicases of two DRH-1 molecules, reminiscent of oligomerization of mammalian RLRs. dsRNA threads through all three helicases with a widened major groove at helicase-helicase interfaces and where the conserved Hel2 loop inserts into the major groove. The presence of nucleotide redistributed helicase-helicase and helicase-RNA contacts in the AVC, as for MDA5, albeit specific interactions and remodeling differed. MDA5 and RIG-I RLRs contain an unstructured linker between their CARDs and helicase domain, but the analogous linker in DRH-1 has a short-structured region positioned to interact with DCR-1. These findings provide a structural framework for understanding how DCR-1, DRH-1, and RDE-4 cooperate to cleave viral dsRNA and elucidate unique features required for antiviral defense in different animals.

immunology↗

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

Detection of Orsay viral replication intermediates reveals spatial and regulatory links to Caenorhabditis elegans innate immune responses

Orsay Virus (OV) is a positive-sense, single-stranded RNA (+ssRNA) virus that naturally infects C. elegans intestines. Like other +ssRNA viruses, the OV-encoded RNA-dependent RNA polymerase (oRdRP) synthesizes complementary antigenome for use as template for amplifying viral genome, but OV replication intermediates are underexplored. Using PCR, we observed viral genome in vast excess of antigenome, as for other +ssRNA viruses. Unlike interferon-based antiviral defense, C. elegans utilizes RNA interference (RNAi) for antiviral defense, producing sense and antisense small interfering RNAs (siRNAs) that cannot be distinguished from genome and antigenome with conventional hybridization protocols. Fluorescence-based imaging in C. elegans intestines using probes to antigenomic sequences revealed cytoplasmic as well as perinuclear localization patterns. The latter depended on factors required for generation of primary, but not secondary, siRNAs, connecting the antigenomic hybridization pattern to RNAi. We also observed cytoplasmic double-stranded RNA (dsRNA) associated with oRdRP, suggesting viral replication hubs, as well as infection-induced nuclear dsRNA, likely from endogenous dsRNA. Finally, using antibodies to oRdRP, we observed spherical structures of [~]1{micro}m in diameter with oRdRP at their surface, which decrease in animals lacking RDE-1. Our study defines features of OV replication intermediates, setting the stage for understanding their connection to host antiviral pathways. Significance StatementO_LIOrsay virus is a +ssRNA virus that infects C. elegans intestines. We advance understanding of viral replication intermediates and address the issue that for animals that use antiviral RNA interference, hybridization of probes occurs with both genome and antigenome and small interfering RNAs. C_LIO_LISingle-molecule fluorescence in-situ hybridization using antigenomic probes revealed cytoplasmic and perinuclear puncta, only upon denaturation, with perinuclear signal dependent on primary, but not secondary, siRNA biogenesis. Viral RNA-dependent RNA polymerase lined the perimeter of spherical structures of [~]1{micro}m diameter. C_LIO_LIThis study sets the stage for understanding the relationship between viral replication dynamics and antiviral RNA interference. C_LI

immunology↗

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↗

bioRxiv2022

Ancestral protein reconstruction reveals evolutionary events governing variation in Dicer helicase function

Antiviral defense in ecdysozoan invertebrates requires Dicer with a helicase domain capable of ATP hydrolysis. But despite well-conserved ATPase motifs, human Dicer is incapable of ATP hydrolysis, consistent with a muted role in antiviral defense. To investigate this enigma, we used ancestral protein reconstruction to resurrect Dicers helicase in animals and trace the evolutionary trajectory of ATP hydrolysis. Biochemical assays indicated ancient Dicer possessed ATPase function, that like extant invertebrate Dicers, is stimulated by dsRNA. Analyses revealed that dsRNA stimulates ATPase activity by increasing ATP affinity, reflected in Michaelis constants. Deuterostome Dicer-1 ancestor, while exhibiting lower dsRNA affinity, retained ATPase activity; importantly, ATPase activity was undetectable in the vertebrate Dicer-1 ancestor, which had even lower dsRNA affinity. Reverting residues in the ATP hydrolysis pocket was insufficient to rescue hydrolysis, but including additional substitutions distant from the ATPase pocket rescued vertebrate Dicer-1s ATPase function. Our work suggests Dicer lost ATPase function in the vertebrate ancestor due to loss of ATP affinity, involving motifs distant from the active site, important for coupling dsRNA binding to the active conformation. RLRs important for interferon signaling, and their competition with Dicer for viral dsRNAs, possibly provided incentive to jettison an active helicase in vertebrate Dicer.

biochemistry↗