The ribosomal P-stalk couples amino acid starvation to GCN2 activation in mammalian cells
Abstract
The eukaryotic translation initiation factor 2α (eIF2α) 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 eIF2α kinase PERK. Wildtype ribosomes isolated from CHO cells, but not those with P-stalk lesions, stimulated GCN2-dependent eIF2α 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.
Additional Information
© 2019 Harding et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Received: 12 July 2019; Accepted: 20 November 2019; Published: 21 November 2019. We thank the CIMR flow cytometry core facility team (Reiner Schulte, Chiara Cossetti and Gabriela Grondys-Kotarba) for help with cell sorting, Miguel Remacha from Centro de Biologia Molecular Severo Ochoa, Madrid, Spain, for the gift of MoAb 3BH5 to the P-stalk, The Lehner lab at CIMR for advice on CRISPR-Cas9 screens, Cambridge CRUK for access to their deep sequencing resource, the Warren lab at CIMR for use of their gradient maker, our colleagues, Claudia Rato, Steffen Preissler, Luke Perera, and Lisa Neidhardt for comments on the manuscript and Claudia Flandoli for the illustrations. This work was supported by Wellcome Trust Principal Research Fellowship to DR (Wellcome 200848/Z/16/Z), a Wellcome Trust Strategic Award to the Cambridge Institute for Medical Research (Wellcome 100140) and Cancer Research UK program grant to RLW (C14801/A21211). Competing interests: David Ron: Reviewing editor, eLife. The other authors declare that no competing interests exist. Author contributions: Heather P Harding, Conceptualization, Formal analysis, Supervision, Investigation, Visualization, Project administration; Adriana Ordonez, Formal analysis, Investigation; Felicity Allen, Leopold Parts, Software, Methodology; Alison J Inglis, Resources, Methodology; Roger L Williams, Resources, Funding acquisition, Methodology; David Ron, Conceptualization, Supervision, Funding acquisition, Investigation, Project administration. Data availability: All data generated or analysed during this study are included in the manuscript, supporting files, or are submitted to public databases.Attached Files
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Supplemental Material - elife-50149-supp1-v2.xlsx
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Additional details
- PMCID
- PMC6919976
- Eprint ID
- 100504
- Resolver ID
- CaltechAUTHORS:20200103-125657310
- 200848/Z/16/Z
- Wellcome Trust
- 100140
- Wellcome Trust
- C14801/A21211
- Cancer Research UK
- Created
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2020-01-05Created from EPrint's datestamp field
- Updated
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2023-10-18Created from EPrint's last_modified field