Transfer RNA acetylation regulates in vivo mammalian stress signaling

Transfer RNA (tRNA) modifications are crucial for protein synthesis, but their position-specific physiological roles remain poorly understood. Here, we investigate the impact of N⁴-acetylcytidine (ac⁴C), a highly conserved tRNA modification catalyzed by the essential acetyltransferase Nat10. By targeting Thumpd1, a nonessential adapter protein required for Nat10-catalyzed tRNA acetylation, we determine that loss of tRNA acetylation leads to reduced levels of tRNA^Leu, increased ribosome stalling, and activation of eIF2α phosphorylation. Thumpd1 knockout mice exhibit growth defects and sterility. Concurrent knockout of Thumpd1 and the stress-sensing kinase Gcn2 causes penetrant postnatal lethality in mice, indicating a critical genetic interaction. Our findings demonstrate that a modification restricted to a single position within type II cytosolic tRNAs can regulate ribosome-mediated stress signaling in mammalian organisms, with implications for our understanding of translational control and therapeutic interventions.