MicroRNAs (miRNAs) are evolutionarily conserved endogenous small non‐coding RNAs that play critical roles in skeletal muscle development. In this study, we identified putative miRNAs that were differentially expressed in the longissimus dorsi muscle between fetus (75 days of pregnancy) and lamb (1 day of age). We detected 1208 miRNAs, 313 of which were differentially expressed. In particular, we found that miR‐145‐5p was differentially and highly expressed in lamb skeletal muscle. In addition, our results demonstrated that overexpression of miR‐145‐5p inhibited the differentiation and apoptosis of goat primary myoblasts (GPMs), whereas knockdown of miR‐145‐5p had the opposite effect. The coding domain sequence (CDS) of ubiquitin‐specific peptidase 13 (USP13) was predicted and validated as a target of miR‐145‐5p. We also demonstrated that the influence as a key regulator of GPMs differentiation is primarily mediated by targeting and inhibiting USP13. Taken together, these results revealed a novel pathway in skeletal muscle development in which miR‐145‐5p targets CDS region of USP13 to regulate differentiation and apoptosis of GPMs.

RUNX1T1 (Runt-related transcription factor 1, translocated to 1) plays a wideranging and diverse role in cellular development, including hematopoiesis and adipogenesis. However, little is known about the function of RUNX1T1 in the skeletal muscle development. Here, we assessed the impact of RUNX1T1 on the proliferation and myogenic differentiation of goat primary myoblasts (GPMs). It was observed that RUNX1T1 is highly expressed during the early stages of myogenic differentiation and the fetal stage. Moreover, the knockdown of RUNX1T1 promotes the proliferation and inhibits myogenic differentiation and mitochondrial biogenesis of GPMs. RNA sequencing analysis revealed that significantly differentially expressed genes in RUNX1T1 knockdown cells were enriched in the calcium signaling pathway. Additionally, we discovered that RUNX1T1 regulates alternative splicing (AS) events involved in myogenesis. We also show that silencing RUNX1T1 blocked the Ca 2+ -CAMK signaling pathway and reduced the expression levels of muscle-specific isoforms of recombinant rho associated coiled coil containing crotein kinase 2 (ROCK2) during myogenic differentiation, partially explaining why RUNX1T1 deficiency leads to the impairment of myotube formation. These findings suggest that RUNX1T1 is a novel regulator of myogenic differentiation that regulates the calcium signaling pathway and AS of ROCK2. Overall, our results highlight the critical role of RUNX1T1 in myogenesis and broaden our understanding of myogenic differentiation.