1. Cell Biology
  2. Genetics and Genomics
Download icon

The ribosomal P-stalk couples amino acid starvation to GCN2 activation in mammalian cells

  1. Heather P Harding  Is a corresponding author
  2. Adriana Ordonez
  3. Felicity Allen
  4. Leopold Parts
  5. Alison J Inglis
  6. Roger L Williams
  7. David Ron  Is a corresponding author
  1. University of Cambridge, United Kingdom
  2. Wellcome Trust Sanger Institute, United Kingdom
  3. Medical Research Council Laboratory of Molecular Biology, United Kingdom
Research Article
  • Cited 0
  • Views 1,154
  • Annotations
Cite this article as: eLife 2019;8:e50149 doi: 10.7554/eLife.50149

Abstract

The eukaryotic translation initiation factor 2a (eIF2a) kinase GCN2 is activated by amino acid starvation to elicit a rectifying physiological program known as the Integrated Stress Response (ISR). A role for uncharged tRNAs as activating ligands of yeast GCN2 is supported experimentally. However, mouse GCN2 activation has recently been observed in circumstances associated with ribosome stalling with no global increase in uncharged tRNAs. We report on a mammalian CHO cell-based CRISPR-Cas9 mutagenesis screen for genes that contribute to ISR activation by amino acid starvation. Disruption of genes encoding components of the ribosome P-stalk, uL10 and P1, selectively attenuated GCN2-mediated ISR activation by amino acid starvation or interference with tRNA charging without affecting the endoplasmic reticulum unfolded protein stress-induced ISR, mediated by the related eIF2a kinase PERK. Wildtype ribosomes isolated from CHO cells, but not those with P-stalk lesions, stimulated GCN2-dependent eIF2a phosphorylation in vitro. These observations support a model whereby lack of a cognate charged tRNA exposes a latent capacity of the ribosome P-stalk to activate GCN2 in cells and help explain the emerging link between ribosome stalling and ISR activation.

Article and author information

Author details

  1. Heather P Harding

    Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom
    For correspondence
    hph23@cam.ac.uk
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7359-7974
  2. Adriana Ordonez

    Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.
  3. Felicity Allen

    Wellcome Trust Sanger Institute, Hinxton, United Kingdom
    Competing interests
    No competing interests declared.
  4. Leopold Parts

    Wellcome Trust Sanger Institute, Hinxton, United Kingdom
    Competing interests
    No competing interests declared.
  5. Alison J Inglis

    Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.
  6. Roger L Williams

    Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7754-4207
  7. David Ron

    Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom
    For correspondence
    dr360@medschl.cam.ac.uk
    Competing interests
    David Ron, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3014-5636

Funding

Cancer Research UK (C14801/A21211)

  • Roger L Williams

Wellcome (Wellcome 100140)

  • David Ron

Wellcome (Wellcome 200848/Z/16/Z)

  • David Ron

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

Reviewing Editor

  1. Nahum Sonenberg, McGill University, Canada

Publication history

  1. Received: July 12, 2019
  2. Accepted: November 20, 2019
  3. Accepted Manuscript published: November 21, 2019 (version 1)

Copyright

© 2019, Harding 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

  • 1,154
    Page views
  • 303
    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)