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.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Raushan K Singh

    Biochemistry, University of Utah, Salt lake City, United States
    For correspondence
    raushan.singh@biochem.utah.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3636-9112
  2. McKenzie Jonely

    Chemistry, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Evan Leslie

    Biochemistry, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Nick A Rejali

    Pathology, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7210-9425
  5. Rodrigo Noriega

    Chemistry, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Brenda L Bass

    Department of Biochemistry, University of Utah, Salt Lake City, United States
    For correspondence
    bbass@biochem.utah.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1728-2254

Funding

National Institute of General Medical Sciences (R01GM121706)

  • Brenda L Bass

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. James M Berger, Johns Hopkins University School of Medicine, United States

Version history

  1. Received: December 16, 2020
  2. Accepted: March 31, 2021
  3. Accepted Manuscript published: March 31, 2021 (version 1)
  4. Version of Record published: April 27, 2021 (version 2)

Copyright

© 2021, Singh et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Raushan K Singh
  2. McKenzie Jonely
  3. Evan Leslie
  4. Nick A Rejali
  5. Rodrigo Noriega
  6. Brenda L Bass
(2021)
Transient kinetic studies of the antiviral Drosophila Dicer-2 reveal roles of ATP in self•nonself discrimination
eLife 10:e65810.
https://doi.org/10.7554/eLife.65810

Share this article

https://doi.org/10.7554/eLife.65810

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    Funding:

    This work was supported by the Weizmann Krenter Foundation and the Weizmann – Ichilov (Tel Aviv Sourasky Medical Center) Collaborative Grant in Biomedical Research, by the Minerva Foundation, by the ISF KillCorona grant 3777/19.