Relevance of N6-methyladenosine regulators for transcriptome: Implications for development and the cardiovascular system

https://doi.org/10.1016/j.yjmcc.2021.05.006Get rights and content

Highlights

  • M6A RNA methylation modulates both coding and non-coding RNAs.

  • A precise and balanced m6A RNA distribution is mandatory for development and cardiovascular homeostasis and disease.

  • M6A has been associated with cardiovascular diseases and it may have clinical relevance as therapeutic target and biomarker.

Abstract

N6-methyladenosine (m6A) is the most abundant and well-studied internal modification of messenger RNAs among the various RNA modifications in eukaryotic cells. Moreover, it is increasingly recognized to regulate non-coding RNAs. The dynamic and reversible nature of m6A is ensured by the precise and coordinated activity of specific proteins able to insert (“write”), bind (“read”) or remove (“erase”) the m6A modification from coding and non-coding RNA molecules. Mounting evidence suggests a pivotal role for m6A in prenatal and postnatal development and cardiovascular pathophysiology. In the present review we summarise and discuss the major functions played by m6A RNA methylation and its components particularly referring to the cardiovascular system. We present the methods used to study m6A and the most abundantly methylated RNA molecules. Finally, we highlight the possible involvement of the m6A mark in cardiovascular disease as well as the need for further studies to better describe the mechanisms of action and the potential therapeutic role of this RNA modification.

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.

References (169)

  • W. Xiao et al.

    Nuclear m(6)A reader YTHDC1 regulates mRNA splicing

    Mol. Cell

    (2016)
  • R.P. Perry et al.

    Existence of methylated messenger RNA in mouse L cells

    Cell

    (1974)
  • C.-M. Wei et al.

    Methylated nucleotides block 5’ terminus of HeLa cell messenger RNA

    Cell

    (1975)
  • U. Schibler et al.

    Comparison of methylated sequences in messenger RNA and heterogeneous nuclear RNA from mouse L cells

    J. Mol. Biol.

    (1977)
  • M. Salditt-Georgieff et al.

    Methyl labeling of HeLa cell hnRNA: a comparison with mRNA

    Cell

    (1976)
  • C.R. Alarcón et al.

    HNRNPA2B1 is a mediator of m(6)A-dependent nuclear RNA processing events

    Cell

    (2015)
  • J.M. Fustin et al.

    RNA-methylation-dependent RNA processing controls the speed of the circadian clock

    Cell

    (2013)
  • S. Lin et al.

    The m(6)A methyltransferase METTL3 promotes translation in human cancer cells

    Mol. Cell

    (2016)
  • K.D. Meyer et al.

    5' UTR m(6)A promotes cap-independent translation

    Cell

    (2015)
  • S. Arslan et al.

    Long non-coding RNAs in the atherosclerotic plaque

    Atherosclerosis

    (2017)
  • A. Shafik et al.

    The emerging epitranscriptomics of long noncoding RNAs

    Biochim. Biophys. Acta

    (2016)
  • P. Boccaletto et al.

    MODOMICS: a database of RNA modification pathways. 2017 update

    Nucleic Acids Res.

    (2018)
  • D. Dominissini et al.

    The dynamic N(1)-methyladenosine methylome in eukaryotic messenger RNA

    Nature

    (2016)
  • K.E. Bohnsack et al.

    Eukaryotic 5-methylcytosine (m(5)C) RNA methyltransferases: mechanisms, cellular functions, and links to disease

    Genes (Basel)

    (2019)
  • J.M. Adams et al.

    Modified nucleosides and bizarre 5'-termini in mouse myeloma mRNA

    Nature

    (1975)
  • D.T. Dubin et al.

    The methylation state of poly A-containing messenger RNA from cultured hamster cells

    Nucleic Acids Res.

    (1975)
  • R. Desrosiers et al.

    Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells

    Proc. Natl. Acad. Sci. U. S. A.

    (1974)
  • G. Jia et al.

    N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO

    Nat. Chem. Biol.

    (2011)
  • C. He

    Grand challenge commentary: RNA epigenetics?

    Nat. Chem. Biol.

    (2010)
  • D. Dominissini et al.

    Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq

    Nature

    (2012)
  • C. Roost et al.

    Structure and thermodynamics of N6-methyladenosine in RNA: a spring-loaded base modification

    J. Am. Chem. Soc.

    (2015)
  • N. Liu et al.

    N(6)-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions

    Nature

    (2015)
  • G. Cao et al.

    Recent advances in dynamic m6A RNA modification

    Open Biol.

    (2016)
  • K.D. Meyer et al.

    Rethinking m(6)A readers, writers, and erasers

    Annu. Rev. Cell Dev. Biol.

    (2017)
  • X. Wang et al.

    Structural basis of N(6)-adenosine methylation by the METTL3-METTL14 complex

    Nature

    (2016)
  • P. Sledz et al.

    Structural insights into the molecular mechanism of the m(6)A writer complex

    Elife

    (2016)
  • A.S. Warda et al.

    Human METTL16 is a N(6)-methyladenosine (m(6)A) methyltransferase that targets pre-mRNAs and various non-coding RNAs

    EMBO Rep.

    (2017)
  • A. Ruszkowska et al.

    Structural insights into the RNA methyltransferase domain of METTL16

    Sci. Rep.

    (2018)
  • X.L. Ping et al.

    Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase

    Cell Res.

    (2014)
  • J. Liu et al.

    A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation

    Nat. Chem. Biol.

    (2014)
  • Y. Yue et al.

    VIRMA mediates preferential m(6)A mRNA methylation in 3'UTR and near stop codon and associates with alternative polyadenylation

    Cell Discov.

    (2018)
  • K.A. Doxtader et al.

    Structural basis for regulation of METTL16, an S-adenosylmethionine homeostasis factor

    Mol. Cell

    (2018)
  • Y. Fu et al.

    FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA

    Nat. Commun.

    (2013)
  • J.S. McTaggart et al.

    FTO is expressed in neurones throughout the brain and its expression is unaltered by fasting

    PLoS One

    (2011)
  • A.J. Ho et al.

    A commonly carried allele of the obesity-related FTO gene is associated with reduced brain volume in the healthy elderly

    Proc. Natl. Acad. Sci. U. S. A.

    (2010)
  • X. Gao et al.

    The fat mass and obesity associated gene FTO functions in the brain to regulate postnatal growth in mice

    PLoS One

    (2010)
  • C. Tang et al.

    ALKBH5-dependent m6A demethylation controls splicing and stability of long 3'-UTR mRNAs in male germ cells

    Proc. Natl. Acad. Sci. U. S. A.

    (2018)
  • P. Gulati et al.

    Fat mass and obesity-related (FTO) shuttles between the nucleus and cytoplasm

    Biosci. Rep.

    (2014)
  • S. Zou et al.

    N(6)-Methyladenosine: a conformational marker that regulates the substrate specificity of human demethylases FTO and ALKBH5

    Sci. Rep.

    (2016)
  • J. Mauer et al.

    Reversible methylation of m(6)Am in the 5' cap controls mRNA stability

    Nature

    (2017)
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    These authors are joint first authors.

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