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

Abstract

Some RIG-I-like receptors 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 RIG-I- like receptors involved in innate immunity.

Structural insights into dsRNA processing by Drosophila Dicer-2–Loqs-PD

“…There are extensive studies on the 5′-triphosphate recognition by the C-terminal domain of RIG-I that has similar conformational change of helicase after dsRNA binding 28 , 29 . Dcr-2 may sense the 5′-triphosphate of RNAs using a similar binding pocket to that demonstrated previously 12 , 27 . Recent single-molecule analysis 19 supports the conclusion from our structures in this study that terminal loading of dsRNA as the initial step and translocation along the dsRNA are required to activate the processive cleavage activity of Dcr-2.…”

“…Recent research suggested that ATP is required for recognition and discrimination of dsRNA termini 12 , 27 . We have identified a 5′-phosphate-binding pocket in Hel2i in the initial binding state (Fig.…”

Structural Insights into the Advances and Mechanistic Understanding of Human Dicer

“…The defining structural difference between Class II and Class III proteins is the addition of an N-terminal duplex RNA-activated ATPase (DRA) helicase domain, also sometimes referred to as a DExD/H-box helicase domain. − The DRA helicase domain, formed by three subdomains, Hel1, Hel2i, and Hel2, binds and unwinds the hairpin loop region of the pre-miRNA substrate. ,− As shown in Figure , while the majority of Dicer and Dicer-like enzymes contain an N-terminal DRA helicase domain, there is variability between species regarding whether this domain is ATP dependent or independent. These differences in ATP dependence are seemingly linked to Dicer’s substrate specificity during cleavage. ,− For instance, in Drosophila , Dicer-1 is ATP independent and primarily cleaves pre-miRNA, while Dicer-2 requires ATP to cleave pre-small interfering RNAs (siRNA). ,− Interestingly, the minimal Giardia Dicer lacks the helicase domain altogether despite being classified as a Class III RNase III enzyme . Lack of a helicase domain may suggest an evolutionary shift in Dicer enzymes requiring a more sophisticated means to differentiate RNA substrates in higher eukaryotes.…”

“…Unlike humans which only contain one copy of Dicer, Drosophila has evolved two copies of Dicer known as Dicer-1 and Dicer-2. , As previously mentioned, these Dicer proteins differ in their ATP dependence with dmDicer-1 cleaving pre-miR substrates independent of ATP, while dmDicer-2 depends on ATP to cleave long dsRNA and discriminate between the dsRNA termini for specific cleavage. ,,, This functional duality is dependent on dmDicer’s helicase domain, but until very recently, we lacked structural insights to explain these activity differences. Using cryo-EM, multiple structures of dmDicer-1 and dmDicer-2 , in a variety of conformational states have been determined ranging in resolution from 5 to 3 Å.…”

“…As previously mentioned, structural studies of dmDicer-1 revealed a high degree of flexibility in the Platform-PAZ domain, but similar conformational flexibility has not been observed for dmDicer-2 , or hsDicer , structures. However, transient kinetic studies of dmDicer-2 do suggest that the Platform-PAZ domain may play a larger role in the initiation of cleavage and stabilization of RNA substrates . To understand the mechanisms for how cleavage sites are regulated and what additional functionals the Platform-PAZ domain might have in hsDicer, additional kinetic and structural investigations that focus on the intermediate steps occurring during hsDicer processing are needed.…”