N6-Methyladenosine

N6-Methyladenosine (m⁶A) is adenosine with a methyl group on the 6-amino group of the adenine base. It is the most prevalent internal modification of mRNA in higher eukaryotes. An early study looked at mRNA from hepatoma cells and found that the nucleosides methylated on the base were mainly N6-methyladenosine. Two transcriptome-wide methods, m⁶A-seq and MeRIP-seq, combine antibody capture of methylated RNA with high-throughput sequencing. They found m⁶A in the mRNAs of thousands of human and mouse genes, with sites concentrated near stop codons.

Formation in RNA. In mammalian cells, m⁶A is added to nuclear RNAs by a methyltransferase complex whose core is a stable heterodimer of METTL3 and METTL14. The methyl group comes from S-adenosylmethionine (SAM). Crystal structures of the METTL3–METTL14 methyltransferase domains show SAM bound in the catalytic site. In one study, the enzymes were given SAM whose methyl group was labelled with deuterium, and the m⁶A they formed carried the label. In mammalian mRNA, methylation takes place within the consensus sequence Pu[G>A]m⁶AC[A/C/U] (Pu = purine), although only some of the sequences that match this pattern carry m⁶A.

Removal by oxidative demethylation. The modification is reversible. In one study, the enzyme FTO removed the methyl group from m⁶A in RNA by oxidation in vitro. In the same study, lowering FTO levels in human cells raised the amount of m⁶A in mRNA, and overexpressing FTO lowered it. A later study reported a second mammalian demethylase, ALKBH5, which removes the methyl group from m⁶A in mRNA by oxidation, both in vitro and in vivo. One study showed that FTO, an Fe(II)- and α-ketoglutarate-dependent enzyme, oxidises m⁶A first to N6-hydroxymethyladenosine and then to N6-formyladenosine, and that both break down to adenosine in water. The same study found both modifications in mRNA isolated from human cells and mouse tissues.

Reader proteins. In one study, the human reader protein YTHDF2 bound m⁶A-containing mRNA through its C-terminal domain. Its N-terminal domain took the bound mRNA from the pool available for translation to sites of mRNA decay, such as processing bodies. m⁶A has been reported to affect the localisation, stability, translation and splicing of mRNA.

Dimroth rearrangement. Under alkaline conditions at elevated temperatures, 1-methyladenosine (m¹A) rearranges to N6-methyladenosine. Because of this, m¹A in RNA may be converted to m⁶A during purification and measurement, which can add noise to m⁶A measurements. One study that mapped m¹A in mRNA used the rearrangement on purpose. m¹A often causes errors during reverse transcription, but m⁶A rarely does, so converting m¹A to m⁶A with alkali helped the authors confirm where m¹A sits.

Modified RNA and immune sensors. In one study, RNA containing m⁶A, made by in vitro transcription, was less able to stimulate the RNA sensor TLR3 and did not stimulate TLR7 or TLR8. These tests used cultured cells engineered to express each human receptor. In the same study, however, RNA containing m⁶A still activated primary dendritic cells from blood as strongly as unmodified RNA. Of the modifications tested, only those on uridine stopped this activation.

Analytical standard. Pure m⁶A has been used, together with pure adenosine, to build standard curves for measuring the m⁶A/A ratio in mRNA by LC-MS/MS.