(A) Overlay plot of the fluorescence signal from wildtype (WT) or GCN2-ablated Eif2ak4∆ mutant HeLa cells with an ISR sensitive CHOP::GFP reporter (horizontal axis Ex: 488 nm/ Em530 ± 30 nm) and a constitutive mCherry reporter (vertical axis Ex 561/Em 610 ± 20 nm). The cells were untreated (UT, red), treated with histidinol (2 mM HeLa, 0.5 mM CHO, HD, blue) or tunicamycin (2 μg/ml, Tm, orange). Histograms of the signal in each channel are displayed on the axes. (B) Flow cytometry plot as in ‘A’ but from wildtype or GCN2-ablated CHO cells with the CHOP::GFP reporter (horizontal axis) and an ER-stress sensitive XBP1::mCherry reporter (vertical axis). Color-coding as in A. (C) Schema used to enrich for cells with CRISPR-Cas9 induced genetic lesions that impair ISR activation (CHOP::GFP dull cells) and for identification of the guide RNA sequences they harbor. (D) Flow cytometry plot as in B but from pools of CRISPR-Cas9 mutagenized CHO cells following two rounds of FACS-based enrichment for histidinol-treated CHOP::GFP dull cells. ‘R2 Med Dull’ and ‘R2 Dullest’ refers to pools selected for medium or strong impairment of CHOP::GFP induction. (E) Plot of the mean log2 fold enrichment or depletion of guides. The vertical axis compares the histidinol-treated CHOP::GFP dull population to an unselected population of histidinol-treated CHO cells. The horizontal axis reports on the enrichment or depletion of guides from unselected cells compared to their abundance in the original library. Guides targeting Eif2ak4, Rplp0, Rplp1 and Rplp2 are color coded. All other ribosomal proteins are in dark grey. Note that only Rplp0 guides that were not depleted from the unselected pool of transduced cells were enriched in the CHOP::GFP dull population. (F) Cartoon of the structure of the human ribosome with the position of the E, P, and A, sites highlighted and the P-stalk (based on PDB 4v6x) in close up. The ribosome associated N-terminal domain (NTD) of uL10 and the P-stalk protrusion are indicated. The unstructured acidic C-termini of uL10, P1, and P2, unresolved in PDB 4v6x, are not shown. The approximate positions on the protein corresponding to the site targeted by the Rplp0 guides enriched in the CHOP::GFP dull cells or depleted from the unselected pool of transduced cells are indicated by the red and grey translucent spheres, respectively.