Transient kinetic studies of the antiviral Drosophila Dicer-2 reveal roles of ATP in self-nonself discrimination

Some RIG-I-like receptors (RLRs) discriminate viral and cellular dsRNA by their termini, and Drosophila melanogaster Dicer-2 (dmDcr-2) differentially processes dsRNA with blunt or 2 nucleotide 3'-overhanging termini. We investigated the transient kinetic mechanism of the dmDcr-2 reaction using a rapid reaction stopped-flow technique and time-resolved fluorescence spectroscopy. Indeed, we found that ATP binding to dmDcr-2's helicase domain impacts association and dissociation kinetics of dsRNA in a termini-dependent manner, revealing termini-dependent discrimination of dsRNA on a biologically relevant time scale (seconds). ATP hydrolysis promotes transient unwinding of dsRNA termini followed by slow rewinding, and directional translocation of the enzyme to the cleavage site. Time-resolved fluorescence anisotropy reveals a nucleotide-dependent modulation in conformational fluctuations (nanoseconds) of the helicase and Platform-PAZ domains that is correlated with termini-dependent dsRNA cleavage. Our study offers a kinetic framework for comparison to other Dicers, as well as all members of the RLRs involved in innate immunity.

Automated summary

This study investigated the transient kinetic mechanism of dmDcr-2 in processing dsRNA, revealing ATP's impact on association and dissociation kinetics in a termini-dependent manner. The study also showed ATP hydrolysis promotes transient unwinding of dsRNA termini, followed by slow rewinding and directional translocation of the enzyme. Time-resolved fluorescence spectroscopy demonstrated nucleotide-dependent modulation in conformational fluctuations of helicase and Platform-PAZ domains correlated with termini-dependent dsRNA cleavage.