N⁶-Methyladenosine

N⁶-Methyladenosine (m⁶A) was originally identified and partially characterised in the 1970s, and is an abundant modification in mRNA and DNA. It is found within some viruses, and most eukaryotes including mammals, insects, plants and yeast. It is also found in tRNA, rRNA, and small nuclear RNA (snRNA) as well as several long non-coding RNA, such as Xist.

The methylation of adenosine is directed by a large m⁶A methyltransferase complex containing METTL3, which is the subunit that binds S-adenosyl-L-methionine (SAM). In vitro, this methyltransferase complex preferentially methylates RNA oligonucleotides containing GGACU and a similar preference was identified in vivo in mapped m⁶A sites in Rous sarcoma virus genomic RNA and in bovine prolactin mRNA. More recent studies have characterized other key components of the m⁶A methyltransferase complex in mammals, including METTL14, Wilms tumor 1 associated protein (WTAP), VIRMA and METTL5. Following a 2010 speculation of m⁶A in mRNA being dynamic and reversible, the discovery of the first m⁶A demethylase, fat mass and obesity-associated protein (FTO) in 2011 confirmed this hypothesis and revitalized the interests in the study of m⁶A. A second m⁶A demethylase alkB homolog 5 (ALKBH5) was later discovered as well.

The biological functions of m⁶A are mediated through a group of RNA binding proteins that specifically recognize the methylated adenosine on RNA. These binding proteins are named m⁶A readers. The YT521-B homology (YTH) domain family of proteins (YTHDF1, YTHDF2, YTHDF3 and YTHDC1) have been characterized as direct m⁶A readers and have a conserved m⁶A-binding pocket. Insulin-like growth factor-2 mRNA-binding proteins 1, 2, and 3 (IGF2BP1–3) are reported as a novel class of m⁶A readers. IGF2BPs use K homology (KH) domains to selectively recognize m6A-containing RNAs and promote their translation and stability. These m⁶A readers, together with m⁶A methyltransferases (writers) and demethylases (erasers), establish a complex mechanism of m⁶A regulation in which writers and erasers determine the distributions of m⁶A on RNA, whereas readers mediate m⁶A-dependent functions. m⁶A has also been shown to mediate a structural switch termed m⁶A switch.

The specificity of m⁶A installation on mRNA is controlled by exon architecture and exon junction complexes. Exon junction complexes suppress m⁶A methylation near exon-exon junctions by packaging nearby RNA and protecting it from methylation by the m⁶A methyltransferase complex. m⁶A regions in long internal and terminal exons, away from exon-exon junctions and exon junction complexes, escape suppression and can be methylated by the methyltransferase complex.