Dynamic Regulation of 5-Formylcytidine on tRNA

Post-transcriptional modifications on RNA play an important role in biological processes, but we lack an understanding of the molecular mechanisms underlying the function of many modifications. Here we characterize the distribution and dynamic regulation of 5-formylcytidine (f⁵C), a modification primarily found on tRNAs, across different cell lines, mouse tissues, and in response to environmental stress. We identify perturbation in bulk f⁵C levels using nucleoside LC-MS and quantify individual modification stoichiometry at the wobble base of mt-tRNA-Met and tRNA-Leu-CAA using nucleotide resolution f⁵C sequencing technology. Our studies show that f⁵C modifications on tRNAs are dynamic, and responsive to fluctuations in cellular iron levels and O₂ concentration. Further, we show using a translation reporter assay that decoding of Leu UUA codons is impaired in cells lacking f⁵C, implicating f⁵C(m)34 on tRNA-Leu-CAA in wobble decoding. Together, our work illuminates dynamic epitranscriptomic mechanisms regulating protein translation in response to environment.