Chemical Method to Sequence 5-Formylcytosine on RNA

Epitranscriptomic RNA modifications can regulate biological processes, but there remains a major gap in our ability to identify and measure individual modifications at nucleotide resolution. Here we present . Mal-Seq, a chemical method for sequencing 5-formylcytosine (f(5)C) modifications on RNA based on the selective and efficient malononitrile-mediated labeling of f(5)C residues to generate adducts that are read as C-to-T mutations upon reverse transcription and polymerase chain reaction amplification. We apply MaI-Seq to characterize the prevalence of f(5)C at the wobble position of mt-tRNA(Met) in different organisms and tissue types and find that high-level f(5)C modification is present in mammals but lacking in lower eukaryotes. Our work sheds light on mitochondrial tRNA modifications throughout eukaryotic evolution and provides a general platform for characterizing the f(5)C epitranscriptome.

Automated summary

Epitranscriptomic RNA modifications play a regulatory role in biological processes. This study presents a chemical method, Mal-Seq, for sequencing 5-formylcytosine (f(5)C) modifications on RNA. The method involves the selective and efficient labeling of f(5)C residues, which are read as C-to-T mutations. The study shows that high-level f(5)C modification is present in mammals but lacking in lower eukaryotes. These findings shed light on mitochondrial tRNA modifications throughout eukaryotic evolution and provide a general platform for characterizing the f(5)C epitranscriptome.