N6-Methyladenosine, or m6A, is formed when a methyl group is chemically added at the nitrogen-6 position of adenosine residues. Often referred to as “the fifth RNA base”, m6A is the most common and abundant eukaryotic RNA modification, accounting for over 80% of all RNA methylation. It can be found mainly in mRNA, but is also observed in non-coding species like tRNA, rRNA, and miRNA.
Through interactions with various binding proteins called “readers”, m6A affects virtually every facet of ribonucleic acid biology: structure, splicing, localization, translation, stability, and turnover [1]. Aside from this central role in RNA metabolism, m6A plays a part in other physiological processes such as cell differentiation, immunity, inflammation, and the circadian clock [2]. Abnormal m6A methylation has been implicated in diabetes, obesity, neurodegeneration, cancer, and other pathologies. The formation of m6A RNA appears to be a co-transcriptional event occurring early on in the RNA lifecycle and is mediated by a multi-protein methyltransferase complex. The recent discoveries of these methylase“writers” (METTL3/14, WTAP, RBM15/15B, VIRMA) and their associated demethylase“erasers” (FTO, ALKBH5, TET-like enzymes) in mammals uncovered the reversibility of the m6A modification, revealing potential therapeutic targets for m6A dysregulation-related diseases.
Reversible m6A methylation in RNA
Epigenetic modifications are also known to influence the life cycles of RNA viruses like human coronavirus. Modified adenosines like m6A are reported to affect the viability of specific RNA viruses by modulating viral cap structures, viral replication, innate sensing pathways, and the innate immune response [3]. Interestingly, members of the coronaviruses encode their own methyltransferases for self-methylating adenosine residues and promoting immune evasion. Thus, examining the effects of m6A methylase/demethylase activity on the epigenome and epitranscriptome of coronaviruses and other RNA viruses, as well as their targets, may provide valuable insights into the impact of viral and host-derived m6A RNA modifications on infection so as to guide remedial courses of action.
Currently, there are few methods available for detecting m6A methylase or demethylase activity using both nuclear extracts and purified enzymes. To address this issue, EpigenTekoffers fastand convenientall-in-one ELISA-based colorimetric assays to rapidly measure activity levels of m6A methylases and demethylasesfrom cell/tissue nuclear extracts or purified enzymes in a high-throughput format. A unique m6A substrate is provided with each kit, which stably binds to the assay wells. Bioactive enzymes from input samples will transfer methyl groups to, or remove them from, the bound substrate, depending on the kit. Substrate m6A methylation is subsequently detected by a high-affinity antibody specific for this modification. Quantitative measurements of methylase/demethylase activity and inhibition (if testing enzyme inhibitors) can be obtained within 4-5 hours. EpigenTek also offers a nuclear extraction kitdesigned to preserve enzymatic activity of extracted nuclear proteins and optimized for use with these ELISA assays.
Hastings MH. m(6)A mRNA methylation: a new circadian pacesetter. Cell. 2013;155(4):740-741. doi:10.1016/j.cell.2013.10.028
Gonzales-van Horn SR, Sarnow P. Making the Mark: The Role of Adenosine Modifications in the Life Cycle of RNA Viruses. Cell Host Microbe. 2017;21(6):661-669. doi:10.1016/j.chom.2017.05.008
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