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

• Published in: eLife Vol. 8
• Main Authors: Harding, Heather P, Ordonez, Adriana, Allen, Felicity, Parts, Leopold, Inglis, Alison J, Williams, Roger L, Ron, David
• Format: Journal Article
• Language: English
• Published: England eLife Science Publications, Ltd 21-11-2019; eLife Sciences Publications Ltd; eLife Sciences Publications, Ltd
• Subjects: Amino acid starvation; Amino acids; Amino Acids - metabolism; Analysis; Animals; Cell activation; Cell Biology; Cells (Biology); CHO; CHO Cells; Cricetulus; CRISPR; CRISPR-Cas Systems; eIF-2 Kinase - genetics; eIF-2 Kinase - metabolism; Endoplasmic reticulum; Endoplasmic Reticulum - metabolism; Flow cytometry; GCN2; Gene expression; Gene Expression Regulation, Enzymologic; Genes; Genetic testing; Genetics and Genomics; Genomes; HeLa Cells; Humans; Initiation factor eIF-2α; integrated stress response; Kinases; Kinetics; Ligands; Mammalian cells; Mice; Models, Molecular; Mutagenesis; Mutation; P-stalk; Phosphorylation; Physiological aspects; Protein Binding; Protein Conformation; Protein folding; Protein Unfolding; Protein-Serine-Threonine Kinases - chemistry; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; Proteins; ribosome stalling; Ribosomes; Ribosomes - chemistry; Ribosomes - metabolism; RNA; RNA, Transfer - metabolism; Shortages; Signal Transduction; Starvation - metabolism; Stress response; Transcriptome; Transfer RNA; Translation (Genetics); tRNA; ul10; United Kingdom; ul10; GCN2; p1; p2; cell biology; genetics; P-stalk; genomics; ribosome stalling; CHO; integrated stress response
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/elife.50149

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.