Background: Arsenic and cadmium are widely distributed in nature and pose serious threats to the environment and human health. Exposure to these nonessential toxic metals may result in a variety of human diseases including cancer. However, arsenic and cadmium toxicity targets and the cellular systems contributing to tolerance acquisition are not fully known.

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In addition to DNA cytosine methylation (5-methyl-2′-deoxycytidine, m5dC), DNA adenine methylation (N6-methyl-2′-deoxyadenosine, m6dA) is another DNA modification that has been discovered in eukaryotes. Recent studies demonstrated that the content and distribution of m6dA in genomic DNA of vertebrates and mammals exhibit dynamic regulation, indicating m6dA may function as a potential epigenetic mark in DNA of eukaryotes besides m5dC. Whether m6dA undergoes the further oxidation in a similar way to m5dC remains elusive. Here, we reported the existence of a new DNA modification, N6-hydroxymethyl-2′-deoxyadenosine (hm6dA), in genomic DNA of mammalian cells and tissues. We found that hm6dA can be formed from the hydroxylation of m6dA by the Fe2+- and 2-oxoglutarate-dependent ALKBH1 protein in genomic DNA of mammals. In addition, the content of hm6dA exhibited significant increase in lung carcinoma tissues. The increased expression of ALKBH1 in lung carcinoma tissues may contribute to the increase of hm6dA in DNA. Taken together, our study reported the existence and formation of hm6dA in genomic DNA of mammals.

The recent discovery of reversible chemical modifications on mRNA has opened a new era of post-transcriptional gene regulation in eukaryotes. Among the 15 types of modifications identified in mRNA of eukaryotes, N7-methylguanosine (mG) is unique owing to its presence in the 5' cap structure. It remains unknown whether mG is also present internally in mRNA, and this is largely attributed to the lack of an appropriate analytical method to differentiate internal mG in mRNA from that in the 5' cap. To address this analytical challenge, we developed a novel strategy of combining differential enzymatic digestion with liquid chromatography-tandem mass spectrometry analysis to quantify the levels of these two types of mG modifications in mRNA. In particular, we found that S1 nuclease and phosphodiesterase I exhibit differential activities toward internal and 5'-terminal mG. By using this method, we found that internal mG was present in mRNA of cultured human cells as well as plants and rat tissue. In addition, our results showed that plants contain higher levels of internal mG in mRNA than mammals. We also observed that exposure of rice to cadmium (Cd) stimulated marked diminution in the levels of mG at both the 5' cap and internal positions of mRNA, which was correlated with the Cd-induced elevated expression of mG-decapping enzymes. Taken together, we reported here a strategy to distinguish internal and 5'-terminal mG in mRNA, and by using this method, we demonstrated the prevalence of internal mG modification in mRNA, which we believe will stimulate future functional studies of mG on post-transcriptional gene regulation in eukaryotes.

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.