Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping

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 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.N4-乙酰胞苷（N4-acetylcytidine，ac4C）是一种古老且高度保守的RNA修饰形式，广泛存在于转运RNA（transfer RNA，tRNA）、核糖体RNA（ribosomal RNA，rRNA）中，近年也在真核生物信使RNA（messenger RNA，mRNA）中得到相关研究。本研究报道了ac4C-seq技术——一种可在全转录组范围内实现单核苷酸分辨率、定量定位ac4C的化学基因组学方法。研究结果显示，人类与酵母的mRNA中未检测到可辨识的ac4C位点；但通过异位过表达真核乙酰转移酶复合物，可诱导产生ac4C修饰，且所有修饰位点均位于保守序列基序内。与之形成鲜明对比的是，跨进化谱系分析表明，超嗜热古菌的核糖体RNA、转运RNA、非编码RNA（non-coding RNA，ncRNA）以及信使RNA中存在数百个ac4C修饰残基，且修饰水平达到前所未有的高度。ac4C的修饰水平会随温度变化显著上调，而乙酰转移酶缺陷型古菌菌株会表现出温度依赖性生长缺陷。通过对野生型（wild type，WT）及乙酰转移酶缺陷型古菌核糖体进行冷冻电镜（cryo-electron microscopy，cryo-EM）可视化分析，为解析ac4C的温度依赖性分布模式及其潜在的热适应功能提供了结构层面的见解。本研究定量界定了ac4C的修饰全景图谱，为阐明该修饰在生物学过程与疾病发生中的作用奠定了技术与概念基础。