The Important Role of m6A-Modified circRNAs in the Differentiation of Intramuscular Adipocytes in Goats Based on MeRIP Sequencing Analysis

Abstract

Intramuscular fat contributes to the improvement of goat meat quality. N⁶-Methyladenosine (m6A)-modified circular RNAs play important roles in adipocyte differentiation and metabolism. However, the mechanisms by which m6A modifies circRNA before and after differentiation of goat intramuscular adipocytes remain poorly understood. Here, we performed methylated RNA immunoprecipitation sequencing (MeRIP-seq) and circRNA sequencing (circRNA-seq) to determine the distinctions in m6A-methylated circRNAs during goat adipocyte differentiation. The profile of m6A-circRNA showed a total of 427 m6A peaks within 403 circRNAs in the intramuscular preadipocytes group, and 428 peaks within 401 circRNAs in the mature adipocytes group. Compared with the intramuscular preadipocytes group, 75 peaks within 75 circRNAs were significantly different in the mature adipocytes group. Furthermore, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of intramuscular preadipocytes and mature adipocytes showed that the differentially m6A-modified circRNAs were enriched in the PKG signaling pathway, endocrine and other factor-regulated calcium reabsorption, lysine degradation, etc. m6A-circRNA-miRNA-mRNA interaction networks predicted the potential m6A-circRNA regulation mechanism in different goat adipocytes. Our results indicate that there is a complicated regulatory relationship between the 12 upregulated and 7 downregulated m6A-circRNAs through 14 and 11 miRNA mediated pathways, respectively. In addition, co-analysis revealed a positive association between m6A abundance and levels of circRNA expression, such as expression levels of circRNA_0873 and circRNA_1161, which showed that m6A may play a vital role in modulating circRNA expression during goat adipocyte differentiation. These results would provide novel information for elucidating the biological functions and regulatory characteristics of m6A-circRNAs in intramuscular adipocyte differentiation and could be helpful for further molecular breeding to improve meat quality in goats.

Keywords: MeRIP-seq (m6A-seq); goat; intramuscular adipocyte; m6A-circRNAs.

Figure 1

Adipocytes differentiated for 3 days. Oil Red O (Scale bars, 50 µm) and BODIPY staining analysis in goat intramuscular adipocyte adipogenesis. Confocal imaging of lipid droplets after staining with 0.2 mM BODIPY 493/503 (green) and DAPI (blue). Data are shown as mean ± SEM, ** p < 0.01. Values for each time point are based on three biological replicates and three technical replicates. IMPA represents intramuscular preadipocytes; IMA represents intramuscular adipocytes.

Figure 2

Schematic diagram of the experimental flow. IMAP indicates intramuscular preadipocytes and IMA indicates intramuscular adipocytes.

Figure 3

Overview of m6A-circRNAs in the IMPA and IMA groups. (A) Venn diagram displaying the specific m6A peaks in the IMPA and IMA groups. (B) Venn diagram showing the specific circRNAs with m6A modification between the two groups. (C) The number of m6A peaks per circRNA in the two groups. (D) The length of m6A-circRNAs in the two groups. (E) The source of m6A-circRNAs in the two groups. (F) Chromosome distribution.

Figure 4

Differential expression of m6A-circRNAs between IMA and IMPA group. (A) Data visualization analysis was performed using IGV, showing the location of differential m6A peaks in the circRNA source genes (circRNA_NUCB1 and ZMYND8) between the IMA group and IMPA group. (B) Heatmap of m6A-circRNA expression per sample. (C) The top 10 GO terms enriched for the source genes of differential m6A-circRNAs in the two groups. (D) The top 10 KEGG pathways enriched for the source genes of differential m6A-circRNAs in the two groups.

Figure 5

m6A-circRNA–miRNA–mRNA ceRNA interaction network. (A) The hypermethylated circRNAs. (B) The hypomethylated circRNAs. The box size represents the strength of the binding. The color indicates that the miRNA has a binding site with mRNA and m6A-circRNA.

Figure 6

Conjoint analysis of circRNAs with or without m6A modification. (A) Volcano plot showing the differentially expressed circRNAs in the IMPA and IMA group. (B) Heatmap illustrating the differentially expressed circRNAs in the two groups. (C) Conjoint analysis of expression levels and m6A modifications in the IMPA and IMA groups.

Figure 7

Comparison of circRNA expressions between real-time PCR and circRNA-seq data. For each circRNA, three technical replicates and three biological replicates were used at each sampling time point.