The cytosolic cysteinyl-tRNA synthetase is capable of regulating ATF4 translation independent of eIF2α phosphorylation state

In mammalian cells, the integrated stress response (ISR) triggers ATF4 translation under general conditions of cell stress. Mammalian target of rapamycin (mTOR) signaling also triggers ATF4 translation under general pro-growth conditions. While the transcriptomic changes of these two contradictory pathways are have been studied, a full understanding of all pathways capable of increasing ATF4 translation and how these pathways are regulated and interact with each other remains unknown. In a genome-wide CRISPRi screen, we found that loss of CARS is sufficient to activate ATF4 translation independent of canonical ISR signaling. This ATF4 translation does not require eIF2α phosphorylation and does not require MTOR kinase activity. In a genome-wide epistasis CRISPRi screen using a staggered sgRNA infection strategy, we identified METAP2 and OGT as potential downstream factors in this pathway. This represents a novel pathway for activation of ATF4 translation that may enable targeted manipulation of ATF4 for beneficial therapeutic outcomes.