Background: Intramuscular preadipocyte differentiation plays a critical role in bovine intramuscular fat (IMF) deposition. However, the roles of different RNAs, including mRNAs, circRNAs, lncRNAs and miRNAs, in regulating the adipogenic differentiation of intramuscular preadipocytes remain largely unclear. Results: In the present study, a whole transcriptome sequencing and analysis, including the analysis of mRNAs, circRNAs, lncRNAs and miRNAs, during different differentiation stages (0, 3, 6, and 9 d) of intramuscular preadipocytes from Qinchuan cattle was performed. All samples were prepared with 3 biological replicates. Here, a total of 27,153 mRNAs, 14,070 circRNAs, 7035 lncRNAs, and 427 miRNAs were annotated. Among them, we identified 4848 differentially expressed mRNAs (DEMs), 181 DE circRNAs (DECs), 501 DE lncRNAs (DELs) and 77 DE miRNAs (DEmiRs) between 0 d and other differentiation days (3, 6, and 9 d). GO and KEGG functional enrichment analyses showed that these differentially expressed genes were mainly enriched in cell differentiation, fat metabolism and adipogenesis-related pathways. Furthermore, weighted gene coexpression network analysis (WGCNA) and co-expression network analysis screened out multiple important mRNAs, circRNAs and lncRNAs related to intramuscular adipogenesis. Based on the competing endogenous RNA (ceRNA) regulatory mechanism, we finally identified 24 potential ceRNA networks and 31 potential key genes, including FOXO1/miR-330/circRNA2018/MSTRG.20301, GPAM/miR-27b/ciRNA489 and SESN3/miR-433/circRNA2627MSTRG.20342. Conclusions: This study provides new insights into the differential expression patterns of different transcript types (i.e., mRNAs, circRNAs, lncRNAs and miRNAs) in intramuscular preadipocyte differentiation. Our findings provide data support for studying the molecular mechanism of key mRNAs and noncoding RNAs in IMF deposition, and provide new candidate markers for the molecular breeding of beef cattle.

N⁶-methyladenosine (m⁶A) plays an important role in regulating gene expression. Previous studies found that m⁶A methylation affects skeletal muscle development. However, the effect of m⁶A methylases on bovine skeletal myogenesis is still unclear. Here, we found that the expression of m⁶A demethylases (FTO and ALKBH5) was significantly higher in the longissimus dorsi muscle of adult cattle than in newborn cattle. In contrast, the expression of m⁶A methyltransferases (METTL3, METTL14 and WTAP) was reduced. The mRNA expression of all five genes was found to be increased during the myogenesis of myoblasts in vitro. Knockdown of FTO or METTL3 promoted myoblast proliferation, inhibited myoblast apoptosis and suppressed myogenic differentiation, whereas ALKBH5 knockdown had the opposite effect. METTL14 knockdown enhanced myoblast proliferation and impaired myogenic differentiation. WTAP knockdown attenuated proliferation and contributed to apoptosis but did not affect differentiation. Furthermore, the functional domains of these five m⁶A methylases are conserved across species. Our results suggest that m⁶A methylases are involved in regulating skeletal muscle development and that there may be a complex network of m⁶A methylation regulating skeletal myogenesis.

Understanding the molecular mechanisms of skeletal myoblast differentiation is essential for studying muscle developmental biology. In our previous study, we reported that knockdown of myocyte enhancer factor 2A (MEF2A) inhibited myoblast differentiation. Here in this study, we further identified that MEF2A controlled this process through regulating the maternally expressed 3 (MEG3)—iodothyronine deiodinase 3 (DIO3) miRNA mega cluster and protein phosphatase 2A (PP2A) signaling. MEF2A was sufficient to induce MEG3 expression in bovine skeletal myoblasts. A subset of miRNAs in the MEG3-DIO3 miRNA cluster was predicted to target PP2A subunit genes. Consistent with these observations, MEF2A regulated PP2A signaling through its subunit gene protein phosphatase 2 regulatory subunit B, gamma (PPP2R2C) during bovine myoblast differentiation. MiR-758 and miR-543 in the MEG3-DIO3 miRNA cluster were down-regulated in MEF2A-depleted myocytes. Expression of miR-758 and miR-543 promoted myoblast differentiation and repressed PPP2R2C expression. Luciferase activity assay showed that PPP2R2C was post-transcriptionally targeted by miR-758 and miR-543. Taken together, these results reveal that the MEG3-DIO3 miRNAs function at downstream of MEF2A to modulate PP2A signaling in bovine myoblast differentiation.

A genome-wide association study (GWAS) is an effective tool for identifying the dominant genes of complex economic traits in livestock by statistical analysis of genotype data and measured phenotype data. In this study, we rigorously measured 14 body conformation traits in 254 Qinchuan cattle, comprising body weight (BW), body height (BOH), back height (BAH), buttock height (BUH), chest depth (CD), chest width (CW), hip cross height (HCH), body length (BL), hip width (HW), rump length (RL), pin bone width (PBW), chest girth (CG), abdomen circumference (AG), and calf circumference (CC). After quality control, 281,889 SNPs were generated for GWAS with different traits. A total of 250 suggestive SNPs (p < 3.54 × 10⁻⁶) were screened and 37 candidate genes were annotated. Furthermore, we performed a linkage disequilibrium analysis of SNP loci and considered published studies, identifying the eight genes (ADAMTS17, ALDH1A3, CHSY1, MAGEL2, MEF2A, SYNM, CNTNAP5, and CTNNA3) most likely to be involved in growth traits. This study provides new insights into the regulatory mechanisms of bovine body size development, which can be very useful in the development of management and breeding strategies.

A crucial goal of reducing backfat thickness (BFT) is to indirectly improve feed conversion efficiency. This phenotype has been reported in certain papers; however, the molecular mechanism has yet to be fully revealed. Two extreme BFT groups, consisting of four Qinchuan cattle, were chosen for this study. We performed metabolite and transcriptome analyses of blood from cattle with a high BFT (H-BFT with average = 1.19) and from those with a low BFT (L-BFT with average = 0.39). In total, 1106 differentially expressed genes (DEGs) and 86 differentially expressed metabolites (DEMs) were identified in the extreme trait. In addition, serum ceramide was strongly correlated with BFT and could be used as a potential biomarker. Moreover, the most notable finding was that the functional genes (SMPD3 and CERS1) and metabolite (sphingosine 1-phosphate (S1P)) were filtered out and significantly enriched in the processes related to the sphingolipid metabolism. This investigation contributed to a better understanding of the subcutaneous fat depots in cattle. In general, our results indicated that the sphingolipid metabolism, involving major metabolites (serum ceramide and S1P) and key genes (SMPD3 and CERS1), could regulate BFT through blood circulation.