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

5-Formylcytidine (f⁵C) is one type of post-transcriptional RNA modification, which is known at the wobble position of tRNA in mitochondria 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, thus inducing C-to-T transition in f⁵C sites during PCR to achieve single-base resolution detection. More than 1000 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.