Dynamic RNA acetylation revealed by cross-evolutionary mapping at base resolution

Aldema Sas-Chen ¹ Justin Thomas ² Donna Matzov ³ Masato Taoka ⁴ Kellie Nance ² Ronit Nir ¹ Keri Bryson ² Ran Shachar ¹ Geraldy Liman ⁵ Brett Burkhart ⁵ Supuni Gamage ² Yuko Nobe ⁴ Chloe Briney ² Michaella Levy ⁶ Ryan Fuchs ⁷ G. Brett Robb ⁷ Jesse Hartmann ¹ Sunny Sharma ⁸ Qishan Lin ⁹ Laurence Florens ⁶ Michael Washburn ⁶ Toshiaki Isobe ⁴ Tom Santangelo ⁵ Moran Shalev-Benami ³ Jordan Meier ² Schraga Schwartz ¹

¹Department of Molecular Genetics, Weizmann Institute of Science, Israel
²National Cancer Institute, National Cancer Institute, USA
³Department of Structural Biology, Weizmann Institute of Science, Israel
⁴Department of Chemistry, Tokyo Metropolitan University, Japan
⁵Department of Biochemistry, Colorado State University, USA
⁶Stowers Institute for Medical Research, Stowers Institute for Medical Research, USA
⁷RNA Research Division, New England Biolabs, USA
⁸Department of Cell Biology and Neuroscience, Rutgers University
⁹RNA Epitranscriptomics and Proteomics Resource, University at Albany, USA

N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification, present on tRNA, rRNA and recently also reported on eukaryotic mRNA. We developed ac4C-seq, a chemical genomic method for single-nucleotide resolution, transcriptome-wide quantitative mapping of ac4C. First, we apply this method to redefine the ac4C landscape in human and yeast. We then conduct a cross-evolutionary profiling which 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 ac4C-deficient strains exhibit temperature-dependent growth defects. Cryo-EM visualization of ac4C at near-atomic resolution in WT and ac4C-deficient archaeal ribosomes furnishes structural insights into the temperature-dependent distribution of this modification and its potential thermoadaptive role. Our studies redefine the ac4C landscapes, and provide the foundations for unravelling the roles of ac4C in health and disease.