The dynamic epitranscriptome: N6-methyladenosine and gene expression control

Abstract

N(6)-methyladenosine (m(6)A) is a modified base that has long been known to be present in non-coding RNAs, ribosomal RNA, polyadenylated RNA and at least one mammalian mRNA. However, our understanding of the prevalence of this modification has been fundamentally redefined by transcriptome-wide m(6)A mapping studies, which have shown that m(6)A is present in a large subset of the transcriptome in specific regions of mRNA. This suggests that mRNA may undergo post-transcriptional methylation to regulate its fate and function, which is analogous to methyl modifications in DNA. Thus, the pattern of methylation constitutes an mRNA 'epitranscriptome'. The identification of adenosine methyltransferases ('writers'), m(6)A demethylating enzymes ('erasers') and m(6)A-binding proteins ('readers') is helping to define cellular pathways for the post-transcriptional regulation of mRNAs.

Figure 1. Spatially distinct pools of m⁶A in mRNAs

Metagene analysis of MeRIP-Seq data shows that m⁶A is enriched in discrete regions of mRNA transcripts. In this approach, each m⁶A peak in the transcriptome was mapped to a virtual transcript based on its position in the mRNA in which it is found. The metagene profile represents an overall frequency distribution of m⁶A residues along the entire mature transcript body. Shown is an idealized metagene profile based on published MeRIP-Seq datasets. m⁶A residues can be found at the first encoded residue ,often as a dimethylated nucleotide (m⁶Am). m⁶A is also found at each of the other indicated positions in mRNAs, with a particularly prominent enrichment near the stop codon in mRNAs.

Figure 2. m⁶A methylation and demethylation pathways

(a) METTL3 and METTL14 are two homologous m⁶A methyltransferases that synergize to methylate adenosines in RNA. WTAP is an additional component of this complex which lacks methyltransferase activity but which has a strong influence on m⁶A formation by interacting with METTL3/14. The central adenosines in the GAC and AAC motifs are the methylation sites of these enzymes. Additional methyltransferases may also contribute to m⁶A formation in cells, such as those which direct N⁶ methylation of adenosines associated with the cap structure. The generation of N⁶-methyladenosine (m⁶A) is shown. (b) FTO and ALKBH5, the two mammalian m⁶A mRNA demethylases identified to date, catalyze methyl group removal from the N⁶ position of adenosine residues. FTO catalyzes oxidation of m6A in a reaction that requires O2, ascorbate, Fe(II) and 2-oxoglutarate. Oxidation of m6A generates CO2, succinate, and Fe(III). The product of this reaction is N⁶-hydroxymethyladenosine, an unstable intermediate that spontaneously decomposes to adenosine and formaldehyde. The squiggly lines refer to the position of the ribose sugar to which the base is attached.

Figure 3. Mechanisms and functions of m⁶A

(a,b) Several mechanisms have been ascribed to m6A. m6A is likely to have a role in facilitating or blocking RNA—protein interactions (a). m6A can also affect RNA by altering RNA structure or folding (b). In standard Watson-Crick base pairing, m⁶A is capable of pairing with uridine. Since a free proton at the N⁶ position is still available for hydrogen bonding, N⁶-methyladenosine behaves like adenosine in its ability to base pair to uridine. The squiggly line refers to the position of the ribose sugar to which the base is attached. m⁶A interferes with the formation of base triples. As is shown in this U•A-U base triple, two free protons are required at the N⁶ position for Hoogsteen base pairing on one face and Watson-Crick base pairing on the other. The presence of a methyl group in place of one of these protons blocks base-triple formation. Thus, m⁶A can disrupt RNA structures dependent on base triples. c-e) Great efforts are underway to determine the effects of m6A on mRNA fate and function. It has been suggested that m6A is targeted to specific intronic regions in order to influence splicing efficiency (c). Adenosine methylation may also lead to increased or reduced translation compared with unmethylated transcripts (d). m6A might promote mRNA degradation by recruiting proteins which target mRNAs toward the cellular degradation machinery. Alternatively, m⁶A might stabilize mRNAs by binding to proteins that promote transcript stability.