In eukaryotes, the use of m6A on mRNA involves a methyltransferase complex, commonly termed the Writer, that installs the methyl group. This m6A modification is recognized by special proteins known as Readers. The number of readers varies across different organisms. Notably, in vertebrates, the presence of proteins categorized as Erasers is suggested to facilitate the removal of m6A, which enables a dynamic regulation of m6A deposition on mRNAs.
The mA mark is added by a mA methyltransferase writer complex post-transcriptionally. This writer complex is composed of METTL3, METTL14, Wilms tumor 1-associated protein (WTAP), KIAA1429 and RBM15. METTL3 is the catalytic subunit, whereas METTL14 is involved in the stability of the complex and RNA recruitment. WTAP is also needed in aiding the recruitment of mRNA, whereas RBM15 and its paralog RBM15B are only involved in the recruitment of lncRNAs. The role RBM15 and RBM15B may have in recruiting other types of RNA to the methyltransferase complex remains unknown. The specific recognition sites of the writers are not known, but the minimal sequence required is 5’-RmAC-3’. METTL3 has been proposed to also be a "reader" of the mA mark. This function is localized in the cytoplasm, where it promotes the recruitment of eIF3. Discovery of the METTL3 complex indicated that m6A installation might be a regulated process, which was pivotal for the advancement and interest in the field of epitranscriptomics.
Members of the YTH domain protein family act as "readers" of mA. The study of these proteins has been key in understanding the functions and effects of mRNA methylation. It has been shown that three members of the human YTH domain family of proteins have higher binding affinities to methylated mRNA. The YTH protein YTHDF2 affects mRNA by directing methylated mRNA from the translational pool to mRNA decay sites. As a result, presence of m6A on mRNA is correlated with a shorter half-life than unmethylated mRNA.
So far, two "erasers" of the mA mark have been identified. ALKBH5 is a demethylase found in mammals that removes the methyl group of mA. The second one is the fat mass and obesity associated protein (FTO), a demethylase that converts mA back to adenosine. FTO preferentially demethylates the mA found closer to the mRNA cap. This oxidative process has three steps and two intermediates: N-hydroxymethyladenosine (hmA) and N-formyladenosine (fA). FTO is most commonly found in nuclear speckles; however, in some species low levels of FTO can also be found in the cytoplasm. Dysfunctional FTO correlates with alterations in body weight and disease, while Alkbh5 knockout mice have impaired fertility. These two facts reflect how important the proper regulation of the mA modification is for normal body function. Moreover, mutations in FTO can lead to developmental failures, brain atrophy and physiological disorders in adulthood.