N⁶-methyladenosine (m⁶A) represents the most abundantly occurring mRNA modification and is involved in the regulation of skeletal muscle development. However, the status and function of m⁶A methylation in prenatal myogenesis remains unclear. In this study, we first demonstrated that knockdown of METTL14, an m⁶A methyltransferase, inhibited the differentiation and promoted the proliferation of C2C12 myoblast cells. Then, using a refined m⁶A-specific methylated RNA immunoprecipitation (RIP) with next generation sequencing (MeRIP-seq) method that is optimal for use with samples containing small amounts of RNA, we performed transcriptome-wide m⁶A profiling for six prenatal skeletal muscle developmental stages spanning two important waves of porcine myogenesis. The results revealed that, along with a continuous decrease in the mRNA expression of the m⁶A reader protein insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), the m⁶A methylome underwent highly dynamic changes across different development stages, with most of the affected genes being enriched in pathways related to skeletal muscle development. RNA immunoprecipitation confirmed that IGF2BP1 targets 76 genes involved in pathways associated with muscle development, including the key marker genes MYH2 and MyoG. Moreover, small interfering RNA (siRNA)-mediated knockdown of IGF2BP1 induced phenotypic changes in C2C12 myoblasts similar to those observed with knockdown of METTL14. In conclusion, we clarified the dynamics of m⁶A methylation and identified key genes involved in the regulatory network of porcine skeletal muscle development.

N ⁶ -甲基腺苷 (m ⁶ A) 代表最丰富的 mRNA 修饰，并参与骨骼肌发育的调节。然而，m ⁶ A 甲基化在产前肌生成中的状态和功能仍不清楚。在这项研究中，我们首先证明了METTL14（一种 m ⁶ A 甲基转移酶）的敲低抑制了分化并促进了 C2C12 成肌细胞的增殖。然后，使用精制的 m ⁶ A 特异性甲基化 RNA 免疫沉淀 (RIP) 和下一代测序 (MeRIP-seq) 方法，该方法最适合用于含有少量 RNA 的样品，我们进行了转录组范围的 m ⁶跨越两个重要的猪肌生成波的六个产前骨骼肌发育阶段的分析。结果表明，与在的mRNA表达的持续下降沿米⁶读者蛋白质胰岛素样生长因子2的mRNA结合蛋白1（IGF2BP1），第m ⁶阿甲基化后行在不同发育阶段的高度动态的变化，大多数受影响的基因在与骨骼肌发育相关的通路中富集。RNA免疫沉淀证实IGF2BP1靶向76个与肌肉发育相关的通路相关的基因，包括关键标记基因MYH2和MyoG. 此外，小干扰 RNA (siRNA) 介导的 IGF2BP1 敲低诱导了 C2C12 成肌细胞的表型变化，类似于 METTL14 敲低所观察到的表型变化。总之，我们阐明了 m ⁶ A 甲基化的动力学，并确定了参与猪骨骼肌发育调控网络的关键基因。