TY - JOUR TI - The ribosomal P-stalk couples amino acid starvation to GCN2 activation in mammalian cells AU - Harding, Heather P AU - Ordonez, Adriana AU - Allen, Felicity AU - Parts, Leopold AU - Inglis, Alison J AU - Williams, Roger L AU - Ron, David A2 - Sonenberg, Nahum A2 - Manley, James L A2 - Sattlegger, Evelyn VL - 8 PY - 2019 DA - 2019/11/21 SP - e50149 C1 - eLife 2019;8:e50149 DO - 10.7554/eLife.50149 UR - https://doi.org/10.7554/eLife.50149 AB - 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. KW - integrated stress response KW - ribosome stalling KW - P-stalk KW - CHO KW - GCN2 KW - ul10 KW - p1 KW - p2 JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -