N6-Methyladenosine (m6A) refers to methylation of the adenosine base at the nitrogen-6 position. The existence of this modification has been known for some time. It is very common in prokaryotic genomes, but has not been found in appreciable amounts in eukaryotic DNA. It is very common however in eukaryotic messenger RNA (mRNA). Studies have shown that approximately 3-5 m6A modifications are found per mRNA in mammals (Narayan and Rottman, 1988; Wei et al., 1975). As interest in various epigenetic marks has increased in recent years, researchers have examined the dynamics and role of n6A in mammals more closely.
Some of the proteins that catalyse the formation, removal, and reading of N6A have recently been elucidated. The fact that n6A has specific methylases and demethylases suggests it is dynamically regulated. The m6A methyltransferase MT-A70 catalyzes the addition of the methyl group onto adenosine. MT-A70 preferentially methylates G(m6A)C and also A(m6A)C to a lesser extent (Wei and Moss, 1977). Two specific demethylases have also been discovered. ALKBH5 oxidizes the N6 methyl group, and this has an impact on RNA metabolism and mRNA export (Zheng et al., 2013). Interestingly, the obesity-associated FTO protein has also been shown to be an n6A demethylase (Jia et al., 2011). The FTO gene was first implicated in obesity using genome-wide association studies. Since this discovery, researchers have been trying to ascertain the function of this putative demethylase. This discovery that it is a n6A mRNA demethylase suggests that n6A plays a significant role in complex phenotypes such as obesity.
Recently, proteins that bind to and “read” n6A have been identified. The YTHDF2 protein has been shown to bind n6A (Wang et al., 2014). YTHDF2-bound RNA relocates to RNA degradation sites within the cell. YTHDF2 has recently been shown to have over 3000 RNA targets, including mostly mRNA but also some non-coding RNAs (Wang et al., 2014). Much more work is needed to understand the full significance of n6A.
m6A Additional Reading
Liu, N., Parisien, M., Dai, Q., Zheng, G., He, C., and Pan, T. (2013). Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA. RNA 19, 1848-1856.
This primary paper presents a method for determining n6A status at single base resolution termed site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET). The authors also go on to use the technique on two lncRNAs and three mRNAs.
This review focuses on recent advances in our understanding of n6A. It covers RNA modifications in general, the dynamics of n6A regulation, and the role of its effector proteins.
- Jia, G., Fu, Y., Zhao, X., Dai, Q., Zheng, G., Yang, Y., Yi, C., Lindahl, T., Pan, T., Yang, Y.G., and He, C. (2011). N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat. Chem. Biol. 7, 885-887.
- Narayan, P., and Rottman, F.M. (1988). An in vitro system for accurate methylation of internal adenosine residues in messenger RNA. Science 242, 1159-1162.
- Wang, X., Lu, Z., Gomez, A., Hon, G.C., Yue, Y., Han, D., Fu, Y., Parisien, M., Dai, Q., Jia, G., et al. (2014). N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117-120.
- Wei, C.M., Gershowitz, A., and Moss, B. (1975). Methylated nucleotides block 5′ terminus of HeLa cell messenger RNA. Cell 4, 379-386.
- Wei, C.M., and Moss, B. (1977). Nucleotide sequences at the N6-methyladenosine sites of HeLa cell messenger ribonucleic acid. Biochemistry 16, 1672-1676.
- Zheng, G., Dahl, J.A., Niu, Y., Fedorcsak, P., Huang, C.M., Li, C.J., Vagbo, C.B., Shi, Y., Wang, W.L., Song, S.H., et al. (2013). ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol. Cell 49, 18-29.