5-Formylcytosine is not a prevalent RNA modification in mammalian cells

Area of Science:

• Biochemistry
• Molecular Biology
• Epigenetics

Background:

• The RNA modification 5-formylcytidine (f5C) is understudied in mammals.
• Previous research suggested low f5C levels in mRNA, potentially due to 5-methylcytidine (m5C) oxidation or demethylation by TET/ALKBH1 enzymes.

Purpose of the Study:

• To accurately quantify and locate f5C in mammalian RNA.
• To investigate the enzymes responsible for f5C generation, particularly TET and ALKBH1.
• To clarify the functional role of f5C in mammalian cells.

Main Methods:

• Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and chemical-assisted sequencing were employed.
• A novel method, FIBo-seq, was developed to enhance specificity and sensitivity for f5C detection.
• Analysis was performed on mouse embryonic stem cells (mESCs).

Main Results:

• The pyridine-borane-sequencing method was found to misidentify other RNA modifications (ac4C) and cytidines as f5C.
• No evidence supports a role for TET enzymes in f5C generation; ALKBH1's role was confirmed.
• f5C was not detected in mRNA; the majority resides in mitochondrial tRNA Methionine (mt-tRNAMet).

Conclusions:

• Mammalian f5C is predominantly located in mt-tRNAMet and is ALKBH1-dependent.
• Previous findings on f5C in mRNA were likely artifacts of sequencing methods.
• f5C likely does not have an instructive function outside of tRNA in mammals.