This comprehensive analysis highlights the critical roles of methyltransferases, demethylases, and binding proteins in RNA modification, along with their implications in physiology and disease.
The Discovery and Prevalence of N6-methyladenosine (m6A)
First discovered in the 1970s, N6-methyladenosine, or m6A, remains the most extensively studied RNA modification. As the most prolific methylation modification in eukaryotic mRNA, m6A has been observed in a wide array of organisms: yeast, plants, insects, mammals, and even viruses. In mammals, this epigenetic modification is present in many different tissues, with the highest expression in the brain, kidney, and liver. It is involved in diverse physiologic processes, including stem cell differentiation, cell division, gametogenesis, and biological rhythms, while aberrancies in m6A methylation have been implicated in assorted complications such as tumors, obesity, and infertility.
Advancements in Understanding m6A Methylation
It was not until a little over a decade ago that scientists gained a better understanding of the modification’s exact location, how it changes over time, and how it is controlled. This progress was forged ahead by the discovery of the first RNA demethylase, fat mass and obesity-associated (FTO) protein [Jia 2011], along with technological advancements like methylated RNA immunoprecipitation and high-throughput sequencing that allowed researchers to accurately map the distribution of m6A across the entire transcriptome.
Addition, deletion, and functionality of the m6A RNA modification
m6A is formed when a methyl group is chemically added at the nitrogen-6 position of adenosine residues. It is a reversible modification that occurs primarily on the A of a highly conserved mRNA sequence known as the RRACH motif, where R can be either G or A (with a preference for G), and H can be U, A, or C (with a preference for U) [Harper 1990]. m6A RNA methylation is regulated by three main types of biomolecules: methyltransferases, or “writers”; demethylases, or “erasers”; and binding proteins, or “readers”, which interact with the m6A-modified RNA to elicit a range of biological functions (Figure 1).
m6A Writers
The methylase that catalyzes m6A RNA formation is a multicomponent complex comprised of the core subunits methyltransferase like 3 (METTL3), METTL14, and Wilms tumor 1-associating protein (WTAP), as well as supplementary components that include vir like m6A methyltransferase associated (VIRMA), RNA binding motif protein 15 (RBM15), and RBM15B. METTL3 can bind to the methyl group donor S-adenosylmethionine (SAM) and catalyze the formation of m6A [Bokar 1997]. It forms a stable complex with METTL14, and the resulting heterodimer can subsequently engage WTAP, which localizes the methyltransferase complex in the nucleus [Ping 2014]. VIRMA, RBM15, and RBM15B have all been reported to interact with WTAP and recruit the complex to target RNAs [Patil 2016; Yue 2018].
m6A Erasers
The m6A demethylases FTO and alkB homolog 5 (ALKBH5) [Zheng 2013] can reverse the m6A methylation process by removing the methyl group from the RNA molecule. Through FTO-mediated oxidative demethylation, m6A is converted in a step-wise manner to N6-hydroximethyladenosine (hm6A) and subsequently N6-formyldenosine (f6A) before finally reverting back to A (Figure 2) [Fu 2013]. Whether such intermediates are generated during m6A demethylation by ALKBH5 requires further investigation [Toh 2020].
m6A Readers
The biofunction of m6A primarily involves post-transcriptional regulation of RNA though interactions with m6A-binding proteins. Of particular note are the YT521-B homology (YTH) N6-methyladenosine RNA binding proteins (YTHDF1-3, YTHDC1-2) and the heterogeneous nuclear ribonucleoproteins (HNRNPA2B1, HNRNPC, HNRNPG). Researchers have discovered that, via these so-called “readers”, m6A affects virtually every facet of ribonucleic acid biology: structure, splicing, localization, translation, stability, and turnover [Zaccara 2019].
YTHDF1 promotes translation of m6A-mRNA by facilitating RNA binding to ribosomes [Wang 2015]. YTHDF2 mediates the degradation of m6A-modified RNAs, including mRNA and some long non-coding RNA [Wang 2014]. YTHDF3 enhances interaction of YTHDF1 and YTHDF2 to their respective RNA substrates, thereby fostering protein synthesis or RNA decay [Shi 2017]. YTHDC1 modulates alternative splicing and nuclear export of m6A-containing mRNA [Xiao 2016; Roundtree 2017], while YTHDC2 enriches the translation efficiency of modified transcripts. HNRNPA2B1, HNRNPC, and HNRNPG have all been identified as regulators of alternative splicing in an m6A-dependent manner [Alarcón 2015; Liu 2015; Liu 2017].
Aside from this central role in RNA metabolism, m6A is a factor in other physiological processes such as cell differentiation, immunity, inflammation, and the circadian clock [Hasting 2013]. Abnormal m6A methylation has been implicated in diverse pathologies: diabetes, obesity, neurodegeneration, and cancer, to name a few. The recent discoveries of m6A methylase “writers” and their associated demethylase “erasers” in mammals uncovered the reversibility of the m6A modification, exposing potential therapeutic targets for m6A dysregulation-related diseases. Continued research in elucidating the dynamics of the m6A RNA methylation machinery, its various components, and the interplay among those components will undoubtedly support the design and development of novel therapies.
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