Widespread, dynamic RNA acetylation as a mechanism for RNA thermostabilization

N4-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 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, which we confirm via biophysical assays. Our studies quantitatively define the ac⁴C landscape, providing a technical and conceptual foundation for unravelling this modification’s role in biology and disease.