Single-Base Resolution Mapping Reveals Distinct 5-Formylcytidine in Saccharomyces cerevisiae mRNAs

5-Formylcytidine (f5C) is one type of post-transcriptional RNA modifi-cations, which is known at the wobble position of tRNA in mitochon-dria and essential for mitochondrial protein synthesis. Here, we show a method to detect f5C modifications in RNA and a transcriptome-wide f5C mapping technique, named f5C-seq. It is developed based on the treatment of pyridine borane, which can reduce f5C to 5,6-dihydrouracil (DHU), thus inducing C-to-T transition in f5C sites during PCR to achieve single-base resolution detection. Thousands of f5C sites were identified after mapping in Saccharomyces cerevisiae by f5C-seq. Moreover, codon composition demonstrated a preference for f5C within wobble sites in mRNA, suggesting the potential role in regulation of translation. These findings expand the scope of the understanding of cytosine modifications in mRNA.5-甲酰基胞嘧啶（5-Formylcytidine, f5C）是一类转录后RNA修饰，已知定位于线粒体tRNA的摆动位点，对线粒体蛋白质合成至关重要。本研究报道了一种用于检测RNA中f5C修饰的方法，以及一种全转录组层面的f5C定位技术——f5C-seq。该技术基于吡啶硼烷处理开发而来，吡啶硼烷可将f5C还原为5,6-二氢尿嘧啶（5,6-dihydrouracil, DHU），进而在聚合酶链式反应（PCR）过程中于f5C位点诱导C到T的碱基转换，从而实现单碱基分辨率的检测。通过f5C-seq对酿酒酵母（Saccharomyces cerevisiae）进行定位分析后，共鉴定到数千个f5C位点。进一步研究发现，密码子组成呈现出mRNA摆动位点偏好f5C修饰的特征，提示其在翻译调控中具有潜在作用。上述发现拓展了学界对mRNA胞嘧啶修饰的认知范畴。