show Abstracthide AbstractN4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification, present on tRNA, rRNA and recently investigated in eukaryotic mRNA. We report ac4C-seq, a chemical genomic method for single-nucleotide resolution, transcriptome-wide quantitative mapping of ac4C. While we did not find detectable ac4C sites in human and yeast mRNAs, ac4C was induced via ectopic overexpression of eukaryotic acetyltransferase complexes, invariably at a conserved sequence motif. In contrast, cross-evolutionary profiling reveals unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, ncRNA and mRNA from hyperthermophilic archaea. Ac4C is dramatically induced in response to temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Cryo-EM visualization of WT and acetyltransferase-deficient archaeal ribosomes furnishes structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for unravelling this modification's role in biology and disease. Overall design: polyA-enriched RNA from HEK-293T WT (single sample) or Nat10+Thumpd1 overexpressing (2 replicates) cells under NaCNBH3, deacetylation or mock treatment. Total RNA from HeLa WT or Nat10 depleted cells (3 replicates each) under NaCNBH3, deacetylation or mock treatment. polyA-enriched RNA from HeLa WT (3 replicates) or Nat10 depleted (2 replicates) cells under NaCNBH3 or mock treatment. Total RNA from M. jannaschiae under NaCNBH3, deacetylation or mock treatment. Total RNA from S. solfataricus under NaCNBH3, deacetylation or mock treatment. Total RNA from T. sp. AM4 under NaCNBH3 or mock treatment, grown in 65, 75 and 85 degrees celsius. Total RNA from P. furiosus under NaCNBH3 or mock treatment, grown in 75, 85 and 95 degrees celsius. Total RNA from T. kodakarensis (T.kod) under NaCNBH3 or mock treatment, grown in 55 (1 sample), 65 (2 samples), 75 (2 samples), 85 (4 samples) and 95 (1 sample) degrees celsius. One of the replicates of 85 degree of T.kod underwent also deacetylation. Total RNA from T. kod strains deleted for TK0754 or TK2097 grown at 85 degrees and treated with NaCMBH3 or mock (in duplicates). T.kod purified ribosomes treated with NaCNBH3. rRNA-depleted RNA from WT T.kod grown at 85 and 95 degrees were treatedd with NaCNBH3 or NaCNBH3 and mock, respectively.