BackgroundThe splicing factor SRSF1 emerges as a mater regulator of cell proliferation, displaying high expression in actively proliferative satellite cells (SCs). In SRSF1 knockout mice (KO) generated via MyoD‐Cre, early mortality and muscle atrophy are observed during postnatal muscle growth. Despite these findings, the precise mechanisms through which SRSF1 loss influences SCs' functions and its role in muscle regeneration remain to be elucidated.MethodsTo unravel the exact mechanisms underlying the impact of SRSF1 deficiency SC functions, we employed single‐cell RNA sequencing (scRNA‐seq) on a mononuclear cell suspension isolated from the newborn diaphragm of KO and control mice. Concurrently, we subjected diaphragm muscles to RNA‐seq analysis to identify dysregulated splicing events associated with SRSF1 deletion. For the analysis of the effect of SRSF1 deletion on muscle regeneration, we generated mice with inducible SC‐specific Srsf1 ablation through Pax7‐CreER. SRSF1 ablation was induced by intraperitoneal injection of tamoxifen. Using cardiotoxin‐induced muscle injury, we examined the consequences of SRSF1 depletion on SC function through HE staining, immunostaining and EdU incorporation assay. C2C12 myoblasts and isolated myoblasts were employed to assess stem cell function and senescence.ResultsUtilizing scRNA‐seq analysis, we observed a noteworthy increase in activated and proliferating myoblasts when SRSF1 was absent. This increase was substantial, with the proportion rising from 28.68% in the control group to 77.06% in the knockout group. However, these myoblasts experienced mitotic abnormalities in the absence of SRSF1, resulting in cell cycle arrest and the onset of cellular senescence. In the knockout mice, the proportion of Pax7+ cells within improper niche positioning increased significantly to 25% compared to 12% in the control cells (n ≥ 10, p < 0.001). Furthermore, there was an observation of persistent cell cycle exit specifically in the Pax7+ cells deficient in SRSF1 (n = 6, p < 0.001). SRSF1 plays a pivotal role in regulating the splicing of Fgfr1op2, favouring the full‐length isoform crucial for mitotic spindle organization. Disrupting SRSF1 in C2C12 and primary myoblasts results in multipolar spindle formation (p < 0.001) and dysregulated splicing of Fgfr1op2 and triggers cellular senescence. Consequently, adult SCs lacking SRSF1 initially activate upon injury but face substantial challenge in proliferation (n = 4, p < 0.001), leading to a failure in muscle regeneration.ConclusionsSRSF1 plays a critical role in SCs by ensuring proper splicing, maintaining mitotic progression and preventing premature senescence. These findings underscore the significant role of SRSF1 in controlling SC proliferation during skeletal muscle growth and regeneration.

中文翻译：

---

SRSF1 对于在骨骼肌生长和再生过程中维持卫星细胞稳态至关重要


背景剪接因子 SRSF1 作为细胞增殖的主调节因子出现，在活跃增殖的卫星细胞 （SCs） 中表现出高表达。在通过 MyoD-Cre 产生的 SRSF1 敲除小鼠 （KO） 中，在出生后肌肉生长过程中观察到早期死亡率和肌肉萎缩。尽管有这些发现，但 SRSF1 缺失影响 SCs 功能及其在肌肉再生中的作用的确切机制仍有待阐明。方法为了揭示 SRSF1 缺陷 SC 功能影响的确切机制，我们对从 KO 和对照小鼠的新生隔膜中分离的单核细胞悬液采用了单细胞 RNA 测序 （scRNA-seq）。同时，我们对膈肌进行 RNA-seq 分析，以确定与 SRSF1 缺失相关的失调剪接事件。为了分析 SRSF1 缺失对肌肉再生的影响，我们通过 Pax7-CreER 生成了具有诱导性 SC 特异性 Srsf1 消融的小鼠。SRSF1 消融是通过腹膜内注射他莫昔芬诱导的。使用心脏毒素诱导的肌肉损伤，我们通过 HE 染色、免疫染色和 EdU 掺入测定检测 SRSF1 耗竭对 SC 功能的影响。采用 C2C12 成肌细胞和分离的成肌细胞评估干细胞功能和衰老。结果利用 scRNA-seq 分析，我们观察到当 SRSF1 不存在时，活化和增殖的成肌细胞显着增加。这种增加是显着的，比例从对照组的 28.68% 上升到基因敲除组的 77.06%。然而，这些成肌细胞在没有 SRSF1 的情况下经历了有丝分裂异常，导致细胞周期停滞和细胞衰老的开始。 在基因敲除小鼠中，不正确的生态位定位中 Pax7 + 细胞的比例显着增加到 25%，而对照细胞为 12% （n ≥ 10，p < 0.001）。此外，在 SRSF1 缺陷的 Pax7 + 细胞中观察到持续细胞周期退出 （n = 6，p < 0.001）。SRSF1 在调节 Fgfr1op2 的剪接中起关键作用，有利于对有丝分裂纺锤体组织至关重要的全长亚型。破坏 C2C12 和原代成肌细胞中的 SRSF1 导致多极纺锤体形成 （p < 0.001） 和 Fgfr1op2 剪接失调并触发细胞衰老。因此，缺乏 SRSF1 的成体 SCs 最初在受伤时激活，但在增殖方面面临重大挑战 （n = 4，p < 0.001），导致肌肉再生失败。结论SRSF1 通过确保正确剪接、维持有丝分裂进程和防止过早衰老，在 SCs 中发挥关键作用。这些发现强调了 SRSF1 在骨骼肌生长和再生过程中控制 SC 增殖的重要作用。