1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
Download icon

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

Research Article
  • Cited 0
  • Views 461
  • Annotations
Cite this article as: eLife 2021;10:e65810 doi: 10.7554/eLife.65810

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.

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

Publication history

  1. Received: December 16, 2020
  2. Accepted: March 31, 2021
  3. Accepted Manuscript published: March 31, 2021 (version 1)

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.

Metrics

  • 461
    Page views
  • 71
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Zachary F Mandell et al.
    Research Article

    NusA and NusG are transcription factors that stimulate RNA polymerase pausing in Bacillus subtilis. While NusA was known to function as an intrinsic termination factor in B. subtilis, the role of NusG in this process was unknown. To examine the individual and combinatorial roles that NusA and NusG play in intrinsic termination, Term-seq was conducted in wild type, NusA depletion, DnusG, and NusA depletion DnusG strains. We determined that NusG functions as an intrinsic termination factor that works alone and cooperatively with NusA to facilitate termination at 88% of the 1400 identified intrinsic terminators. Our results indicate that NusG stimulates a sequence-specific pause that assists in the completion of suboptimal terminator hairpins with weak terminal A-U and G-U base pairs at the bottom of the stem. Loss of NusA and NusG leads to global misregulation of gene expression and loss of NusG results in flagella and swimming motility defects.

    1. Biochemistry and Chemical Biology
    Vidyasiri Vemulapalli et al.
    Research Article Updated

    SHP2 is a protein tyrosine phosphatase that normally potentiates intracellular signaling by growth factors, antigen receptors, and some cytokines, yet is frequently mutated in human cancer. Here, we examine the role of SHP2 in the responses of breast cancer cells to EGF by monitoring phosphoproteome dynamics when SHP2 is allosterically inhibited by SHP099. The dynamics of phosphotyrosine abundance at more than 400 tyrosine residues reveal six distinct response signatures following SHP099 treatment and washout. Remarkably, in addition to newly identified substrate sites on proteins such as occludin, ARHGAP35, and PLCγ2, another class of sites shows reduced phosphotyrosine abundance upon SHP2 inhibition. Sites of decreased phospho-abundance are enriched on proteins with two nearby phosphotyrosine residues, which can be directly protected from dephosphorylation by the paired SH2 domains of SHP2 itself. These findings highlight the distinct roles of the scaffolding and catalytic activities of SHP2 in effecting a transmembrane signaling response.