Active demethylation of 5-methylcytosine (5mC) can be realized through ten-eleven translocation (TET) dioxygenase-mediated oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), followed by thymine DNA glycosylase (TDG)-initiated base excision repair (BER). The TDG-BER pathway may lead to the generation of DNA strand breaks, potentially compromising genome integrity. Alternatively, direct decarboxylation of TET-produced 5caC is highly attractive because this mechanism allows for conversion of 5mC to cytosine without the formation of DNA strand breaks. However, cleavage of the C-C bond in 5caC in human cells remains an open question. We examined this reaction in cell extract and live cells using 5caC-carrying hairpin DNA substrate. After incubation with whole-cell protein extract or transfection into human cells, we monitored the transformation of 5caC to cytosine through direct decarboxylation or BER using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses at both the mononucleotide and oligodeoxynucleotide levels. Our results clearly showed the direct conversion of 5caC to cytosine in human cells, providing evidence to support a novel pathway for active DNA demethylation.

The discovery of dynamic and reversible modifications in messenger RNA is opening new directions in RNA modification-mediated regulation of biological processes.

RNA contains diverse modifications that exert important influences in a variety of cellular processes. So far more than 150 modifications have been identified in various RNA species, mainly in rRNA and tRNA. Recent research advances in RNA modifications have been sparked by the discovery of dynamic and reversible modifications in mRNA. Moving beyond the abundant tRNA and rRNA to mRNA is opening new directions in understanding RNA modification-mediated regulation of gene expression. Recently, it was reported that N 3 -methylcytidine (m 3 C) existed in mRNA of mammalian cells, and methyltransferase-like 8 (METTL8) was identified to be the writer enzyme of m 3 C. However, little is known about the eraser enzyme of m 3 C in mRNA. In the current study, we found that the AlkB homologue 1 (ALKBH1) was capable of demethylating m 3 C in mRNA of mammalian cells in vitro. Overexpression and knockdown of ALKBH1 in cultured human cells can induce decrease and increase of the level of m 3 C in mRNA, respectively, revealing the eraser enzyme property of ALKBH1 on m 3 C in mRNA. In addition, we observed significant decrease of the level of m 3 C in mRNA in hepatocellular carcinoma (HCC) tissues compared to tumor-adjacent normal tissues, which could be attributed to the increased expression of ALKBH1 as well as the decreased expression of METTL8 in HCC tissues. These results indicated that m 3 C in mRNA may play certain roles in tumorigenesis. Our study shed light on understanding the demethylation of m 3 C in mRNA.

DNA cytosine methylation (5-methylcytosine, 5mC) is the most important epigenetic mark in higher eukaryotes. 5mC in genomes is dynamically controlled by the writers and erasers. DNA (cytosine-5)-methyltransferases (DNMTs) are responsible...

The discovery of 5-hydroxymethylcytosine (5hmC) in mammalian genomes is a landmark in epigenomics study. Similar to 5-methylcytosine (5mC), 5hmC is viewed a critical epigenetic modification. Deciphering the functions of 5hmC...