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.
Caenorhabditis elegans Proteins , Caenorhabditis elegans , RNA, Double-Stranded , Ribonuclease III , Animals , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , RNA, Double-Stranded/metabolism , Ribonuclease III/metabolism , Ribonuclease III/chemistry , Ribonuclease III/genetics , Cryoelectron Microscopy , DEAD-box RNA Helicases/metabolism , DEAD-box RNA Helicases/chemistry , DEAD-box RNA Helicases/genetics , RNA Helicases/metabolism , RNA Helicases/genetics , RNA Helicases/chemistry , Protein Binding , Adenosine Triphosphate/metabolism , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , DEAD Box Protein 58/metabolism , DEAD Box Protein 58/genetics , DEAD Box Protein 58/chemistry
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.
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 Dicer's 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 some 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 additional substitutions distant from the pocket rescued vertebrate Dicer-1's 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. By competing with Dicer for viral dsRNA, RIG-I-like receptors important for interferon signaling may have allowed or actively caused loss of ATPase function.
Biological Evolution , RNA, Double-Stranded , Animals , Humans , RNA, Double-Stranded/genetics , Ribonuclease III/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , DNA Helicases/metabolism , Antiviral Agents , Adenosine Triphosphate/metabolism
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.
