N6-methyl-adenosine (m6A) in RNA: an old modification with a novel epigenetic function

Abstract

N(6)-methyl-adenosine (m(6)A) is one of the most common and abundant modifications on RNA molecules present in eukaryotes. However, the biological significance of m(6)A methylation remains largely unknown. Several independent lines of evidence suggest that the dynamic regulation of m(6)A may have a profound impact on gene expression regulation. The m(6)A modification is catalyzed by an unidentified methyltransferase complex containing at least one subunit methyltransferase like 3 (METTL3). m(6)A modification on messenger RNAs (mRNAs) mainly occurs in the exonic regions and 3'-untranslated region (3'-UTR) as revealed by high-throughput m(6)A-seq. One significant advance in m(6)A research is the recent discovery of the first two m(6)A RNA demethylases fat mass and obesity-associated (FTO) gene and ALKBH5, which catalyze m(6)A demethylation in an α-ketoglutarate (α-KG)- and Fe(2+)-dependent manner. Recent studies in model organisms demonstrate that METTL3, FTO and ALKBH5 play important roles in many biological processes, ranging from development and metabolism to fertility. Moreover, perturbation of activities of these enzymes leads to the disturbed expression of thousands of genes at the cellular level, implicating a regulatory role of m(6)A in RNA metabolism. Given the vital roles of DNA and histone methylations in epigenetic regulation of basic life processes in mammals, the dynamic and reversible chemical m(6)A modification on RNA may also serve as a novel epigenetic marker of profound biological significances.

Figure 1

Reversible m⁶A methylation in mRNA METTL3-containing methyltransferase complex catalyzes m⁶A methylation with SAM as a methyl donor. FTO and ALKBH5 demethylate m⁶A in an iron and α-ketoglutarate-dependent manner.

Figure 2

Schematic functional domains of m⁶A methyltransferases and demethylases A. Structural alignment of human METTL3 and its homologues in S. cerevisiae, D. melanogaster, and A. thaliana. Orange box represents motif I, which is a potential Adomet binding domain. Red box represents motif II, which is a potential catalytic domain. Individual protein size (aa) and position of functional domains are shown as indicated. B. Structural alignment of m⁶A demethylases. Both FTO and ALKBH5 belong to the AlkB family. Individual protein size (aa) and positions of conserved iron-binding domain, substrates and α-ketoglutarate interaction motifs are shown as indicated.

Figure 3

Working model of m⁶A in regulating mRNA metabolism As a ubiquitous modification in mRNAs, m⁶A methylation occurs immediately after pre-mRNA transcription by METTL3-containing methyltransferases; while FTO and ALKBH5 are responsible for m⁶A demethylation. Through mutual interplay between methyltransferases and demethylases, m⁶A level is kept in balance to form a docking site for m⁶A binding proteins and proper assembly of RNA secondary structure. The mRNA transcripts with adequate m⁶A level can be properly spliced, transported, translated or degraded. Unbalanced m⁶A regulation will cause defects in RNA metabolism at each step shown above. Functional studies on methyltransferase, demethylases and potential m⁶A-binding proteins reveal that m⁶A in RNA may be involved in adipogenesis (FTO), spermatogenesis (ALKBH5), development (METTL3, FTO and ALKBH5), carcinogenesis (YTHDF2), stem cell renewal and other unidentified life processes.