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 23
  • Views 3,754
  • Annotations
Cite this article as: eLife 2019;8:e50149 doi: 10.7554/eLife.50149
Voice your concerns about research culture and research communication: Have your say in our 7th annual survey.

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.

Data availability

All data generated or analysed during this study are included in the manuscript, supporting files, or are submitted to public data bases.

The following data sets were generated

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)
  4. Version of Record published: December 18, 2019 (version 2)

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

  • 3,754
    Page views
  • 731
    Downloads
  • 23
    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. Cell Biology
    Na Li et al.
    Research Article Updated

    Adiponectin is essential for the regulation of tissue substrate utilization and systemic insulin sensitivity. Clinical studies have suggested a positive association of circulating adiponectin with healthspan and lifespan. However, the direct effects of adiponectin on promoting healthspan and lifespan remain unexplored. Here, we are using an adiponectin null mouse and a transgenic adiponectin overexpression model. We directly assessed the effects of circulating adiponectin on the aging process and found that adiponectin null mice display exacerbated age-related glucose and lipid metabolism disorders. Moreover, adiponectin null mice have a significantly shortened lifespan on both chow and high-fat diet. In contrast, a transgenic mouse model with elevated circulating adiponectin levels has a dramatically improved systemic insulin sensitivity, reduced age-related tissue inflammation and fibrosis, and a prolonged healthspan and median lifespan. These results support a role of adiponectin as an essential regulator for healthspan and lifespan.

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics
    Axel F Brilot et al.
    Research Article

    Microtubule (MT) nucleation is regulated by the γ-tubulin ring complex (γTuRC), conserved from yeast to humans. In Saccharomyces cerevisiae, γTuRC is composed of seven identical γ-tubulin small complex (γTuSC) sub-assemblies, which associate helically to template MT growth. γTuRC assembly provides a key point of regulation for the MT cytoskeleton. Here, we combine crosslinking mass spectrometry, X-ray crystallography, and cryo-EM structures of both monomeric and dimeric γTuSCs, and open and closed helical γTuRC assemblies in complex with Spc110p to elucidate the mechanisms of γTuRC assembly. γTuRC assembly is substantially aided by the evolutionarily conserved CM1 motif in Spc110p spanning a pair of adjacent γTuSCs. By providing the highest resolution and most complete views of any γTuSC assembly, our structures allow phosphorylation sites to be mapped, surprisingly suggesting that they are mostly inhibitory. A comparison of our structures with the CM1 binding site in the human γTuRC structure at the interface between GCP2 and GCP6 allows for the interpretation of significant structural changes arising from CM1 helix binding to metazoan γTuRC.