Dynamic m6A mRNA Methylation Reveals the Role of METTL3/14-m6A-MNK2-ERK Signaling Axis in Skeletal Muscle Differentiation and Regeneration

N6-methyladenosine (m6A) RNA methylation has emerged as an important factor in various biological processes by regulating gene expression. However, the dynamic profile, function and underlying molecular mechanism of m6A modification during skeletal myogenesis remain elusive. Here, we report that members of the m6A core methyltransferase complex, METTL3 and METTL14, are downregulated during skeletal muscle development. Overexpression of either METTL3 or METTL14 dramatically blocks myotubes formation. Correspondingly, knockdown of METTL3 or METTL14 accelerates the differentiation of skeletal muscle cells. Genome-wide transcriptome analysis suggests ERK/MAPK is the downstream signaling pathway that is regulated to the greatest extent by METTL3/METTL14. Indeed, METTL3/METTL14 expression facilitates ERK/MAPK signaling. Via MeRIP-seq, we found that MNK2, a critical regulator of ERK/MAPK signaling, is m6A modified and is a direct target of METTL3/METTL14. We further revealed that YTHDF1 is a potential reader of m6A on MNK2, regulating MNK2 protein levels without affecting mRNA levels. Furthermore, we discovered that METTL3/14-MNK2 axis was up-regulated notably after acute skeletal muscle injury. Collectively, our studies revealed that the m6A writers METTL3/METTL14 and the m6A reader YTHDF1 orchestrate MNK2 expression posttranscriptionally and thus control ERK signaling, which is required for the maintenance of muscle myogenesis and may contribute to regeneration.

[1]  L. Qu,et al.  PERK Signaling Controls Myoblast Differentiation by Regulating MicroRNA Networks , 2021, Frontiers in Cell and Developmental Biology.

[2]  Q. Xiong,et al.  METTL3-Mediated m6A Methylation Regulates Muscle Stem Cells and Muscle Regeneration by Notch Signaling Pathway , 2021, Stem cells international.

[3]  Shu-juan Xie,et al.  METTL3 regulates skeletal muscle specific miRNAs at both transcriptional and post-transcriptional levels. , 2021, Biochemical and biophysical research communications.

[4]  Shu-juan Xie,et al.  N-methyladenine demethylase ALKBH1 inhibits the differentiation of skeletal muscle. , 2021, Experimental cell research.

[5]  Shu-juan Xie,et al.  The functional analysis of transiently upregulated miR-101 suggests a “braking” regulatory mechanism during myogenesis , 2021, Science China Life Sciences.

[6]  P. Carmeliet,et al.  Macrophage-derived glutamine boosts satellite cells and muscle regeneration , 2020, Nature.

[7]  O. Malysheva,et al.  A defined N6-methyladenosine (m6A) profile conferred by METTL3 regulates muscle stem cell/myoblast state transitions , 2020, Cell Death Discovery.

[8]  O. Malysheva,et al.  A defined N6-methyladenosine (m6A) profile conferred by METTL3 regulates muscle stem cell/myoblast state transitions. , 2020, Cell death discovery.

[9]  Yalan Yang,et al.  Longitudinal epitranscriptome profiling reveals the crucial role of N6-methyladenosine methylation in porcine prenatal skeletal muscle development. , 2020, Journal of genetics and genomics = Yi chuan xue bao.

[10]  Lin Qi,et al.  Multifaceted Functions and Novel Insight Into the Regulatory Role of RNA N6-Methyladenosine Modification in Musculoskeletal Disorders , 2020, Frontiers in Cell and Developmental Biology.

[11]  S. Zhang,et al.  Roles of N6-Methyladenosine (m6A) in Stem Cell Fate Decisions and Early Embryonic Development in Mammals , 2020, Frontiers in Cell and Developmental Biology.

[12]  Liwei Xie,et al.  METTL3 is essential for postnatal development of brown adipose tissue and energy expenditure in mice , 2020, Nature Communications.

[13]  Y. Kuang,et al.  Hypoxia Promotes Vascular Smooth Muscle Cell (VSMC) Differentiation of Adipose-Derived Stem Cell (ADSC) by Regulating Mettl3 and Paracrine Factors , 2020, Stem cells international.

[14]  Chuan He,et al.  Where, When, and How: Context-Dependent Functions of RNA Methylation Writers, Readers, and Erasers. , 2019, Molecular cell.

[15]  Lijia Ma,et al.  Mettl3-mediated mRNA m6A methylation promotes dendritic cell activation , 2019, Nature Communications.

[16]  M. Ferrer,et al.  MEK inhibition induces MYOG and remodels super-enhancers in RAS-driven rhabdomyosarcoma , 2018, Science Translational Medicine.

[17]  Wei Li,et al.  METTL3-mediated m6A modification is required for cerebellar development , 2018, PLoS biology.

[18]  Yu-Sheng Chen,et al.  Dynamic transcriptomic m6A decoration: writers, erasers, readers and functions in RNA metabolism , 2018, Cell Research.

[19]  Jianjun Chen,et al.  RNA N6-methyladenosine modification in cancers: current status and perspectives , 2018, Cell Research.

[20]  Mark Helm,et al.  Zc3h13/Flacc is required for adenosine methylation by bridging the mRNA-binding factor Rbm15/Spenito to the m6A machinery component Wtap/Fl(2)d , 2018, Genes & development.

[21]  Hui Zhou,et al.  Inhibition of the JNK/MAPK signaling pathway by myogenesis-associated miRNAs is required for skeletal muscle development , 2018, Cell Death & Differentiation.

[22]  Stefan Hüttelmaier,et al.  Recognition of RNA N6-methyladenosine by IGF2BP Proteins Enhances mRNA Stability and Translation , 2018, Nature Cell Biology.

[23]  Yue Sheng,et al.  METTL14 Inhibits Hematopoietic Stem/Progenitor Differentiation and Promotes Leukemogenesis via mRNA m6A Modification. , 2017, Cell stem cell.

[24]  M. Bühler,et al.  Zc 3 h 13 / Flacc is required for adenosine methylation by bridging the mRNA binding factor Rbm 15 / Spenito to the m 6 A machinery component Wtap / Fl ( 2 ) , 2018 .

[25]  Samie R Jaffrey,et al.  Rethinking m6A Readers, Writers, and Erasers. , 2017, Annual review of cell and developmental biology.

[26]  E. Oki,et al.  The requirement of Mettl3-promoted MyoD mRNA maintenance in proliferative myoblasts for skeletal muscle differentiation , 2017, Open Biology.

[27]  Tao Pan,et al.  Dynamic RNA Modifications in Gene Expression Regulation , 2017, Cell.

[28]  Xiawei Wei,et al.  FTO is required for myogenesis by positively regulating mTOR-PGC-1α pathway-mediated mitochondria biogenesis , 2017, Cell Death & Disease.

[29]  Ping Wang,et al.  Structural Basis for Cooperative Function of Mettl3 and Mettl14 Methyltransferases. , 2016, Molecular cell.

[30]  C. Stewart,et al.  Regenerative function of immune system: Modulation of muscle stem cells , 2016, Ageing Research Reviews.

[31]  X. Liu,et al.  Discovery of a BTK/MNK dual inhibitor for lymphoma and leukemia , 2016, Leukemia.

[32]  Shuye Tian,et al.  The MAP kinase-interacting kinases regulate cell migration, vimentin expression and eIF4E/CYFIP1 binding. , 2015, The Biochemical journal.

[33]  S. Tavazoie,et al.  N6-methyladenosine marks primary microRNAs for processing , 2015, Nature.

[34]  Qi Zhou,et al.  m(6)A RNA methylation is regulated by microRNAs and promotes reprogramming to pluripotency. , 2015, Cell stem cell.

[35]  Yun-Gui Yang,et al.  Dynamic m6A modification and its emerging regulatory role in mRNA splicing , 2015 .

[36]  Chuan He,et al.  FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis , 2014, Cell Research.

[37]  Heather A. Harrington,et al.  Nuclear to cytoplasmic shuttling of ERK promotes differentiation of muscle stem/progenitor cells , 2014, Development.

[38]  T. Ørntoft,et al.  Mnk2 alternative splicing modulates the p38-MAPK pathway and impacts Ras-induced transformation. , 2014, Cell reports.

[39]  Samir Adhikari,et al.  Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase , 2014, Cell Research.

[40]  O. Elemento,et al.  Comprehensive Analysis of mRNA Methylation Reveals Enrichment in 3′ UTRs and near Stop Codons , 2012, Cell.

[41]  Sherry Chin,et al.  MNK2 Inhibits eIF4G Activation Through a Pathway Involving Serine-Arginine–Rich Protein Kinase in Skeletal Muscle , 2012, Science Signaling.

[42]  Hui Zhou,et al.  Insulin-Like Growth Factor-1 Receptor Is Regulated by microRNA-133 during Skeletal Myogenesis , 2011, PloS one.

[43]  T. Mak,et al.  Combined deficiency for MAP kinase-interacting kinase 1 and 2 (Mnk1 and Mnk2) delays tumor development , 2010, Proceedings of the National Academy of Sciences.

[44]  C. Proud,et al.  Features of the Catalytic Domains and C Termini of the MAPK Signal-integrating Kinases Mnk1 and Mnk2 Determine Their Differing Activities and Regulatory Properties* , 2005, Journal of Biological Chemistry.

[45]  S. Nagata,et al.  Mnk2 and Mnk1 Are Essential for Constitutive and Inducible Phosphorylation of Eukaryotic Initiation Factor 4E but Not for Cell Growth or Development , 2004, Molecular and Cellular Biology.

[46]  Jonathan A. Cooper,et al.  Mitogen‐activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2 , 1997, The EMBO journal.