NCAPG is a subunit of condensin I that plays a crucial role in chromatin condensation during mitosis. NCAPG has been demonstrated to be associated with farm animal growth traits. However, its role in regulating myoblast differentiation is still unclear. We used myoblasts derived from fetal bovine tissue as an in vitro model and found that NCAPG was expressed during myogenic differentiation in the cytoplasm and nucleus. Silencing NCAPG prolonged the mitosis and impaired the differentiation due to increased myoblast apoptosis. After 1.5 days of differentiation, silencing NCAPG enhanced muscle-specific gene expression. An assay for transposase-accessible chromatin- high throughput sequencing (ATAC-seq) revealed that silencing NCAPG altered chromatin accessibility to activating protein 1 (AP-1) and its subunits. Knocking down the expression of the AP-1 subunits fos-related antigen 2 (FOSL2) or junB proto-oncogene (JUNB) enhanced part of the muscle-specific gene expression. In conclusion, our data provide valuable evidence about NCAPG’s function in myogenesis, as well as its potential role in gene expression.

MicroRNAs modulate a variety of cellular events, including skeletal muscle development, but the molecular basis of their functions in fetal bovine skeletal muscle development is poorly understood. In this study, we report that bta-miR-24-3p promotes the myogenic differentiation of fetal bovine PDGFRα^- progenitor cells. The expression of bta-miR-24-3p increased during myogenic differentiation. Overexpression of bta-miR-24-3p significantly promoted myogenic differentiation, but inhibited proliferation. A dual-luciferase assay identified ACVR1B as a direct target of bta-miR-24-3p. Similarly, knocking down ACVR1B by RNA interference also significantly inhibited proliferation and promoted the differentiation of bovine PDGFRα^- progenitor cells. Thus, our study provides a mechanism in which bta-miR-24-3p regulates myogenesis by inhibiting ACVR1B expression.

Intramuscular fat (IMF) is one of the major factors determining beef quality. IMF formation is influenced by multiple conditions including genetic background, age and nutrition. In our previous investigation, bta-miR-210 was found to be increased during adipogenesis using miRNA-seq. In this study, we validated the upregulation of bta-miR-210 in platelet-derived growth factor receptor α positive (PDGFRα⁺) progenitor cells during adipogenic differentiation in vitro. To investigate its role in adipogenesis, bta-miR-210 mimics were introduced into progenitor cells, which resulted in enhanced intracellular lipid accumulation. Accordingly, the expression of adipocyte-specific genes significantly increased in the bta-miR-210 mimic group compared to that in the negative control group (p < 0.01). Dual-luciferase reporter assays revealed that WISP2 is a target of bta-miR-210. WISP2 knockdown enhanced adipogenesis. In conclusion, bta-miR-210 positively regulates the adipogenesis of PDGFRα⁺ cells derived from bovine fetal muscle by targeting WISP2.

MiR-23a, a member of the miR-23a/24-2/27a cluster, has been demonstrated to play pivotal roles in many cellular activities. However, the mechanisms of how bta-miR-23a controls the myogenic differentiation (MD) of PDGFRα⁻ bovine progenitor cells (bPCs) remain poorly understood. In the present work, bta-miR-23a expression was increased during the MD of ^PDGFRα− bPCs. Moreover, bta-miR-23a overexpression significantly promoted the MD of ^PDGFRα− bPCs. Luciferase reporter assays showed that the 3’-UTR region of MDFIC (MyoD family inhibitor domain containing) could be a promising target of bta-miR-23a, which resulted in its post-transcriptional down-regulation. Additionally, the knockdown of MDFIC by siRNA facilitated the MD of ^PDGFRα− bPCs, while the overexpression of MDFIC inhibited the activating effect of bta-miR-23a during MD. Of note, MDFIC might function through the interaction between MyoG transcription factor and MEF2C promoter. This study reveals that bta-miR-23a can promote the MD of ^PDGFRα− bPCs through post-transcriptional downregulation of MDFIC.

As organisms develop, individual cells generate mitochondria to fulfill physiologic requirements. However, it remains unknown how mitochondrial network expansion is scaled to cell growth and impacted by environmental cues. The mitochondrial unfolded protein response (UPR ^mt ) is a signaling pathway mediated by the transcription factor ATFS-1 which harbors a mitochondrial targeting sequence (MTS) ¹ . Here, we demonstrate that ATFS-1 mediates an adaptable mitochondrial expansion program that is active throughout normal development. Developmental mitochondrial network expansion required the relatively inefficient MTS ² in ATFS-1, which allowed the transcription factor to be responsive to parameters that impact protein import capacity of the entire mitochondrial network. Increasing the strength of the ATFS-1 MTS impaired UPR ^mt activity throughout development due to increased accumulation within mitochondria. The insulin-like signaling-TORC1 ³ and AMPK pathways affected UPR ^mt activation ^4,5 in a manner that correlated with protein synthesis. Manipulation to increase protein synthesis caused UPR ^mt activation. Alternatively, S6 kinase inhibition had the opposite effect due to increased mitochondrial accumulation of ATFS-1. However, ATFS-1 with a dysfunctional MTS ⁶ constitutively increased UPR ^mt activity independent of TORC1 function. Lastly, expression of a single protein with a strong MTS, was sufficient to expand the muscle cell mitochondrial network in an ATFS-1-dependent manner. We propose that mitochondrial network expansion during development is an emergent property of the synthesis of highly expressed mitochondrial proteins that exclude ATFS-1 from mitochondrial import, causing UPR ^mt activation. Mitochondrial network expansion is attenuated once ATFS-1 can be imported.

The accumulation of deleterious mitochondrial genomes (ΔmtDNAs) underlies inherited mitochondrial diseases and contributes to the aging-associated decline in mitochondrial function. In heteroplasmic cells, oxidative phosphorylation (OXPHOS) function declines as the population of ΔmtDNAs increase relative to wildtype mtDNAs. In response to mitochondrial perturbations, the bZIP protein ATFS-1 induces a transcription program to promote the recovery of mitochondrial function. Paradoxically, ATFS-1 is also required to maintain ΔmtDNAs in heteroplasmic worms. However, the mechanism(s) by which ATFS-1 promotes ΔmtDNA accumulation relative to wildtype mtDNAs is unclear. Here, we show that mitochondrial-localized ATFS-1 binds almost exclusively to ΔmtDNAs in heteroplasmic worms. Moreover, we demonstrate that mitochondrial ATFS-1 promotes the preferential binding of the mtDNA replicative polymerase (POLG) to ΔmtDNAs. Interestingly, inhibition of the mtDNA-bound protease LONP-1 increased ATFS-1 and POLG binding to wildtype mtDNAs. Furthermore, LONP-1 inhibition in C. elegans and human cybrid cells improved the heteroplasmy ratio and restored OXPHOS function. Our findings suggest that ATFS-1 promotes mtDNA replication by recruiting POLG to mtDNA in a manner that is antagonized by LONP-1. We speculate that this mechanism promotes the repair and expansion of the mitochondrial network by synchronizing mtDNA replication with UPR ^mt activation driven by nuclear ATFS-1 activity. However, this repair mechanism cannot resolve OXPHOS defects in mitochondria harboring ΔmtDNAs, resulting in an accumulation of ATFS-1 in dysfunctional mitochondria and constitutive replication of ΔmtDNAs.