TY  - JOUR
AU  - Dehnen, Jasmin A
AU  - Gopanenko, Alexander V
AU  - Scholz, Carola
AU  - Musheev, Michael U
AU  - Niehrs, Christof
TI  - 5-Formylcytosine is not a prevalent RNA modification in mammalian cells.
JO  - Nature Communications
VL  - 16
IS  - 1
SN  - 2041-1723
CY  - [London]
PB  - Springer Nature
M1  - DKFZ-2025-02422
SP  - 9925
PY  - 2025
N1  - #LA:A050# /  DKFZ-ZMBH Alliance
AB  - 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.
KW  - Animals
KW  - Mice
KW  - Cytosine: analogs & derivatives
KW  - Cytosine: metabolism
KW  - Cytidine: analogs & derivatives
KW  - Cytidine: metabolism
KW  - AlkB Homolog 1, Histone H2a Dioxygenase: metabolism
KW  - AlkB Homolog 1, Histone H2a Dioxygenase: genetics
KW  - Mouse Embryonic Stem Cells: metabolism
KW  - RNA, Messenger: metabolism
KW  - RNA, Messenger: genetics
KW  - RNA, Transfer, Met: metabolism
KW  - RNA, Transfer, Met: genetics
KW  - Proto-Oncogene Proteins: metabolism
KW  - Proto-Oncogene Proteins: genetics
KW  - RNA: metabolism
KW  - RNA Processing, Post-Transcriptional
KW  - Tandem Mass Spectrometry
KW  - DNA-Binding Proteins
KW  - Cytosine (NLM Chemicals)
KW  - Cytidine (NLM Chemicals)
KW  - 5-formylcytosine (NLM Chemicals)
KW  - AlkB Homolog 1, Histone H2a Dioxygenase (NLM Chemicals)
KW  - RNA, Messenger (NLM Chemicals)
KW  - RNA, Transfer, Met (NLM Chemicals)
KW  - Alkbh1 protein, mouse (NLM Chemicals)
KW  - Proto-Oncogene Proteins (NLM Chemicals)
KW  - RNA (NLM Chemicals)
KW  - N-acetylcytidine (NLM Chemicals)
KW  - TET1 protein, mouse (NLM Chemicals)
KW  - DNA-Binding Proteins (NLM Chemicals)
LB  - PUB:(DE-HGF)16
C6  - pmid:41219180
C2  - pmc:PMC12606350
DO  - DOI:10.1038/s41467-025-66090-3
UR  - https://inrepo02.dkfz.de/record/306182
ER  -