Table_1_Dynamic m6A mRNA Methylation Reveals the Role of METTL3/14-m6A-MNK2-ERK Signaling Axis in Skeletal Muscle Differentiation and Regeneration.XLSX

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.

N6-甲基腺嘌呤（N6-methyladenosine，m6A）RNA甲基化通过调控基因表达，已成为多种生物学过程中的关键调控因子。然而，在骨骼肌生成过程中，m6A修饰的动态特征、生物学功能及潜在分子机制仍未明确。本研究发现，m6A核心甲基转移酶复合物的成员METTL3与METTL14在骨骼肌发育过程中表达下调。单独过表达METTL3或METTL14均可显著抑制肌管形成。与之相对，敲低METTL3或METTL14则会促进骨骼肌细胞的分化。全基因组转录组分析结果显示，ERK/MAPK信号通路是受METTL3/METTL14调控最为显著的下游信号通路。后续实验证实，METTL3/METTL14的表达可增强ERK/MAPK信号通路的活性。通过甲基化RNA免疫沉淀测序（MeRIP-seq），我们发现ERK/MAPK信号通路的关键调控因子MNK2存在m6A修饰，且是METTL3/METTL14的直接靶标基因。进一步研究表明，YTHDF1是MNK2上m6A修饰的潜在识别蛋白，可在不改变MNK2 mRNA水平的前提下调控其蛋白质表达量。此外，我们发现急性骨骼肌损伤后，METTL3/14-MNK2调控轴的表达水平显著上调。综上，本研究证实，m6A写入酶（writer）METTL3/METTL14与m6A读取蛋白（reader）YTHDF1通过转录后调控MNK2的表达，进而调控ERK信号通路；这一过程对维持骨骼肌生成不可或缺，且可能参与肌肉损伤后的修复再生。