%0 Journal Article
%A Dehnen, Jasmin A
%A Gopanenko, Alexander V
%A Scholz, Carola
%A Musheev, Michael U
%A Niehrs, Christof
%T 5-Formylcytosine is not a prevalent RNA modification in mammalian cells.
%J Nature Communications
%V 16
%N 1
%@ 2041-1723
%C [London]
%I Springer Nature
%M DKFZ-2025-02422
%P 9925
%D 2025
%Z #LA:A050# /  DKFZ-ZMBH Alliance
%X The RNA modification 5-formylcytidine (f5C) is poorly explored in mammals. Low f5C levels reported in mRNA may reflect spurious 5-methylcytidine (m5C) oxidation or targeted demethylation by TET or ALKBH1 dioxygenases. We analyzed f5C in RNA of mouse embryonic stem cells (mESCs) using LC-MS/MS and chemical-assisted sequencing. We reveal that the previously reported pyridine-borane-sequencing misidentifies N4-acetylcytidine (ac4C) and unmodified, hyper-reactive cytidines in a CUMC context as f5C. To overcome these limitations, we developed FIBo-seq with enhanced specificity and sensitivity for f5C-sequencing. We find no evidence for a role of TET enzymes in generating f5C, unlike for ALKBH1. Moreover, no f5C sites are detectable in mRNA. Instead, the bulk of mammalian f5C resides in the well-established mitochondrial tRNA Methionine (mt-tRNAMet) and is mediated by ALKBH1. The results argue against an instructive function for f5C outside tRNA in mammals.
%K Animals
%K Mice
%K Cytosine: analogs & derivatives
%K Cytosine: metabolism
%K Cytidine: analogs & derivatives
%K Cytidine: metabolism
%K AlkB Homolog 1, Histone H2a Dioxygenase: metabolism
%K AlkB Homolog 1, Histone H2a Dioxygenase: genetics
%K Mouse Embryonic Stem Cells: metabolism
%K RNA, Messenger: metabolism
%K RNA, Messenger: genetics
%K RNA, Transfer, Met: metabolism
%K RNA, Transfer, Met: genetics
%K Proto-Oncogene Proteins: metabolism
%K Proto-Oncogene Proteins: genetics
%K RNA: metabolism
%K RNA Processing, Post-Transcriptional
%K Tandem Mass Spectrometry
%K DNA-Binding Proteins
%K Cytosine (NLM Chemicals)
%K Cytidine (NLM Chemicals)
%K 5-formylcytosine (NLM Chemicals)
%K AlkB Homolog 1, Histone H2a Dioxygenase (NLM Chemicals)
%K RNA, Messenger (NLM Chemicals)
%K RNA, Transfer, Met (NLM Chemicals)
%K Alkbh1 protein, mouse (NLM Chemicals)
%K Proto-Oncogene Proteins (NLM Chemicals)
%K RNA (NLM Chemicals)
%K N-acetylcytidine (NLM Chemicals)
%K TET1 protein, mouse (NLM Chemicals)
%K DNA-Binding Proteins (NLM Chemicals)
%F PUB:(DE-HGF)16
%9 Journal Article
%$ pmid:41219180
%2 pmc:PMC12606350
%R 10.1038/s41467-025-66090-3
%U https://inrepo02.dkfz.de/record/306182