Relevance of N6-methyladenosine regulators for transcriptome: Implications for development and the cardiovascular system
Graphical abstract
Introduction
RNA modifications are types of co-transcriptional and/or posttranscriptional regulations that can affect stability, translation and degradation of RNA molecules. Indeed, to date 172 RNA modifications have been identified and reported in the MODOMICS database, among which 72 include methyl groups [1]. Methyl modifications can be found in all types of RNA: messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small and long non-coding RNA (lncRNA). The biological functions of the various RNA modifications change widely based on the biogenesis, the RNA molecule targeted, and the specific nucleotide modified. For instance, N1-methyladenosine (m1A) modification, which is mainly found in tRNA and mRNA, alters the Watson-Crick base pairing and creates a positive electrostatic charge on the modified adenosine. This positive charge can dramatically alter RNA secondary structures, critical for tRNA function [2], and protein-RNA interactions [3]. 5-methylcytosine (m5C) is another RNA modification occurring in the carbon 5 of cytosine. This modification is found in several species of RNA (tRNA, rRNA, mRNA and non-coding RNA) where it seems to play important roles in the regulation of gene expression [4].
N6-methyladenosine (m6A) is the most abundant internal modification observed in mRNAs and lncRNAs in eukaryotes. Although having been first identified in the 1970s [[5], [6], [7], [8]], interest in the biological relevance of this modification was rekindled in recent years as a result of two main advancements: 1) the identification of the first demethylase enzyme, fat-mass and obesity-associated protein (FTO), which confirmed that m6A is indeed dynamic and reversible [9], and thus can be implicated in regulatory processes [10]; 2) the development of high-throughput methods which allowed for the mapping of m6A sites in both mRNAs and lncRNAs [11,12]. The genome-wide profiling of m6A offered the first view of the m6A epitranscriptome. Indeed, m6A generally occurs in the highly conserved RNA consensus motif DRACH (D = A/G/U; R = A/G; H = A/U/C) and exhibits preferential enrichment within pre-mRNA internal long exons, 3’UTR or around the stop codon [11,12].
M6A is able to alter the structure of the target RNA by forcing the rotation of the methylamino group to an anti-conformation position, destabilizing the thermodynamics of the RNA duplex by 0.5-1.7 kcal/mol [13]. The structural changes occurring in the target RNA makes it accessible to the binding of RNA binding proteins in a mechanism called “the m6A switch” [14].
Specific proteins are able to insert (“writers”), bind (“readers”) or remove (“erasers”) m6A in a dynamic manner, determining the abundance and functions of the m6A mark. For both coding and non-coding RNAs, dynamic modifications represent a new layer of control of genetic information that affect stability, translation or splicing processes [[15], [16], [17]].
Section snippets
The m6A regulatory machinery
The m6A “writers” are methyltransferases that insert the m6A modification on RNA molecules (Fig. 1). These enzymes form a multicomponent complex composed of a methyltransferase-like 3 and -14 (METTL3 and METTL14) heterodimer, methyltransferase-like16 (METTL16), Wilm’s tumor 1 associated protein (WTAP) and vir like m6A methyltransferase associated (VIRMA). METTL3 has been established as the primary catalytic component of this complex, whereas its homologue METTL14 is essential for the allosteric
m6A detection methods
Despite the discovery of m6A RNA methylation in the 1970s [8,[60], [61], [62], [63], [64]], its functions were poorly understood until the recent emergence of several high-throughput sequencing methods allowing for the transcriptome wide analysis of the m6A modification (Fig. 2). In 2012, Dominissini et al. and Meyer et al. reported the first transcriptome-wide mapping of m6A sites in individual RNAs [11,12]. The techniques called m6A-seq and MeRIPSeq, respectively, are based on random RNA
m6A in mRNAs
It has been proposed that the deposition of the m6A mark on mRNA transcripts serves to shape the outcome of gene expression through the modulation of multiple steps in the mRNA metabolic process, from nuclear maturation to translation and eventual decay [78].
The m6A RNA methylation process has been linked to the regulation of early mRNA processing, with initial studies proposing m6A to function as a regulator of splicing. This was based on the observations that m6A was more abundant in nuclear
m6A associated enzymes in development
Mounting evidence has revealed a crucial role for m6A RNA methylation in embryonic development and stem cell differentiation. Studies by Wang et al. report the loss of self-renewal capabilities of mouse embryonic stem cells (mESCs) following the knock down of METTL3 and METTL14 [115]. MESCs depleted of METTL3 or METTL14 also exhibited a downregulation of most pluripotency factors, including SOX2, DPPA3 and NANOG, paralleled by an increase in developmental regulators. Under normal developmental
Implications of m6A regulators in cardiac homeostasis and disease
Despite growing interest in the biological significance of m6A RNA methylation, its involvement in the modulation of cardiovascular homeostasis and disease is only recently beginning to be understood (Table 1). This section of the review will provide an overview of the role of m6A RNA methylation and it’s regulatory components in cardiovascular disease, this topic has also been summarised in recent reviews by Qin et al., and Wu et al. [124,125]. Dorn et al, elucidated the relevance of METTL3
Roles of m6A regulators in the vasculature
Recent studies have described the roles of m6A in the regulation of several known vasoactive factors in the context of cancer cell biology. This section of the review will not address these findings, instead we will focus on recent studies investigating the relevance of m6A in the vasculature and in endothelial cell (EC) biology (Table 1)
The first definitive haematopoietic stem and progenitor cells (HSPCs) are directly produced from the hemogenic endothelium during embryogenesis in a process
m6A in cardiovascular disease risk factors
A single nucleotide polymorphism (SNP) within FTO (rs9939609 T>A) has been associated with an increased susceptibility to coronary heart disease (CHD) in two Swedish population-based case-control studies [146]. A subsequent study conducted in a Pakistani cohort corroborated the association of the FTO SNP rs9939609 with coronary artery disease (CAD) and obesity [147]. A study also investigated the impact of the FTO rs9939609 variant on cardiovascular events and related deaths in a 19-year follow
Concluding remarks and translational perspectives
The reversible and dynamic nature of m6A associated with the abundance and short half-life of RNA molecules emphasizes the central role played by this epitranscriptomic modification in different cellular processes. Indeed, cellular m6A homeostasis is ensured by the coordinated activity of m6A “writers” and “erasers”. It is becoming apparent that disrupting this homeostatic state plays a pivotal role in disease development and progression.
Even though, in the recent years many studies have been
Funding
This work is funded by British Heart Foundation Programme Grant and Personal Chair Awards (RG/15/5/31446 and CH/15/1/31199 to CE), Diabetes UK (Diabetes UK 16/0005564 to CE, supporting the PhD studentship of W.K.S and Diabetes UK 18/0005874 grant to A.C.), Luxemburg National Research Fund (grants # C14/BM/8225223, COVID-19/2020-1/14719577/miRCOVID to Y.D. and C17/BM/11613033 to Y.D., supporting the PhD studentship of F.M.S.), Luxemburg Ministry of Higher Education and Research (to Y.D.),
Declaration of Competing Interest
None.
Acknowledgments
This article is based upon work from EU-CardioRNA COST Action CA17129 (www.cardiorna.eu) supported by COST (European Cooperation in Science and Technology).
We thank Dr Pilar Ruiz-Lozano (Imperial College London) for her help in preparing the graphical abstract.
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These authors are joint first authors.