Muscle-specific adult stem cells (MuSCs) are required for skeletal muscle regeneration. To ensure efficient skeletal muscle regeneration after injury, MuSCs must undergo state transitions as they are activated from quiescence, give rise to a population of proliferating myoblasts, and continue either to terminal differentiation, to repair or replace damaged myofibers, or self-renewal to repopulate the quiescent population. Changes in MuSC/myoblast state are accompanied by dramatic shifts in their transcriptional profile. Previous reports in other adult stem cell systems have identified alterations in the most abundant internal mRNA modification, N6-methyladenosine (m⁶A), conferred by its active writer, METTL3, to regulate cell state transitions through alterations in the transcriptional profile of these cells. Our objective was to determine if m⁶A-modification deposition via METTL3 is a regulator of MuSC/myoblast state transitions in vitro and in vivo. Using liquid chromatography/mass spectrometry we identified that global m⁶A levels increase during the early stages of skeletal muscle regeneration, in vivo, and decline when C2C12 myoblasts transition from proliferation to differentiation, in vitro. Using m⁶A-specific RNA-sequencing (MeRIP-seq), a distinct profile of m⁶A-modification was identified, distinguishing proliferating from differentiating C2C12 myoblasts. RNAi studies show that reducing levels of METTL3, the active m⁶A methyltransferase, reduced global m⁶A levels and forced C2C12 myoblasts to prematurely differentiate. Reducing levels of METTL3 in primary mouse MuSCs prior to transplantation enhanced their engraftment capacity upon primary transplantation, however their capacity for serial transplantation was lost. In conclusion, METTL3 regulates m⁶A levels in MuSCs/myoblasts and controls the transition of MuSCs/myoblasts to different cell states. Furthermore, the first transcriptome wide map of m⁶A-modifications in proliferating and differentiating C2C12 myoblasts is provided and reveals a number of genes that may regulate MuSC/myoblast state transitions which had not been previously identified.

骨骼肌再生需要肌肉特异性成体干细胞 (MuSC)。为了确保受伤后骨骼肌的有效再生，MuSC 在从静止状态激活时必须经历状态转换，产生大量增殖的成肌细胞，并继续终末分化，修复或替换受损的肌纤维，或自我更新以重新填充静止人口。 MuSC/成肌细胞状态的变化伴随着其转录谱的巨大变化。先前在其他成体干细胞系统中的报告已经确定了最丰富的内部 mRNA 修饰 N6-甲基腺苷 (m ⁶ A) 的改变，该修饰由其活跃的写入者 METTL3 赋予，通过改变这些细胞的转录谱来调节细胞状态转换。我们的目的是确定通过 METTL3 的 m ⁶ A 修饰沉积是否是体外和体内 MuSC/成肌细胞状态转变的调节剂。使用液相色谱/质谱分析，我们发现在体内骨骼肌再生的早期阶段，整体 m ⁶ A 水平增加，而在体外，当 C2C12 成肌细胞从增殖转变为分化时，整体 m 6 A 水平下降。使用 m ⁶ A 特异性 RNA 测序 (MeRIP-seq)，鉴定了 m ⁶ A 修饰的独特特征，区分了增殖与分化的 C2C12 成肌细胞。 RNAi 研究表明，降低 METTL3（活性 m ⁶ A 甲基转移酶）的水平会降低整体 m ⁶ A 水平，并迫使 C2C12 成肌细胞过早分化。 移植前降低原代小鼠 MuSC 中 METTL3 的水平可增强其在原代移植时的植入能力，但其连续移植的能力会丧失。总之，METTL3 调节 MuSC/成肌细胞中的 m ⁶ A 水平，并控制 MuSC/成肌细胞向不同细胞状态的转变。此外，提供了增殖和分化的 C2C12 成肌细胞中 m ⁶ A 修饰的第一个转录组全图谱，并揭示了许多可能调节 MuSC/成肌细胞状态转变的基因，而这些基因先前尚未鉴定。