m6A Methylation Mediated Autophagy and Nucleotide-Binding Oligomerization Domain-like Receptors Signaling Pathway Provides New Insight into the Mitigation of Oxidative Damage by Mulberry Leaf Polysaccharides

Abstract

m⁶A methylation modification is an important genetic modification involved in biological processes such as sexual maturation, antibacterial, and antiviral in aquatic animals. However, few studies have been conducted in aquatic animals on the relationship between m⁶A methylation modification and autophagy-inflammation induced by lipid metabolism disorders. In the present study, a high-fat (HF) group and HF-MLP group (1 g mulberry leaf polysaccharides (MLPs)/1 kg HF diet) were set up. The mid-hind intestines of Megalobrama amblycephala juveniles from the two groups were collected for MeRIP-seq and RNA-seq after an 8-week feeding trial. The m⁶A peaks in the HF and HF-MLP groups were mainly enriched in the 3' Untranslated Region (3'UTR), Stop codon, and coding sequence (CDS) region. Compared with the HF group, the m⁶A peaks in the HF-MLP group were shifted toward the 5'UTR region. 'RRACH' was the common m⁶A methylation motif in the HF and HF-MLP groups. Methyltransferase mettl14 and wtap expression in the intestines of the HF-MLP group were significantly higher compared with the HF group (p < 0.05). A total of 21 differentially expressed genes(DEGs) with different peaks were screened by the combined MeRIP-seq and RNA-seq analysis. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis enriched BCL2 interacting protein 3 (bnip3) to autophagy-animal and mitophagy-animal signaling pathways, etc., and nucleotide-binding domain leucine-rich repeat protein 1 (nlrp1) was enriched to the Nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway. Combined MeRIP-seq and RNA-seq analysis indicated that the expression pattern of bnip3 was hyper-up and that of nlrp1 was hyper-down. Gene Set Enrichment Analysis (GSEA) analysis confirmed that the intestinal genes of HF-MLP group positively regulate lysosomal and autophagy-animal signaling pathways. In the present study, we demonstrated that m⁶A methylation modification plays a role in regulating autophagy-inflammatory responses induced by HF diets by MLPs, and further explored the molecular mechanisms by which MLPs work from the epigenetic perspective.

Keywords: Megalobrama amblycephala; autophagy; m6A methylation; mulberry leaf polysaccharides; transcriptome.

Figure 1

MLPs altered m⁶A modification in the intestines of HF and HF-MLP groups. (A) Classification of methylated genes in the intestines of the HF and HF-MLP groups. (B–D) Peak density analysis and classification of m⁶A methylation modification sites in intestinal mRNA of the HF and HF-MLP groups. (E) Circos plot of the levels of m⁶A peaks and expression abundance on 24 chromosomes in the HF and HF-MLP groups. (F) GO categorization analysis of genes with differentially methylated m⁶A peaks. (G) KEGG pathway analysis of genes with differential methylated m⁶A peaks. (H) The same motif which was identified in the intestines of the HF and HF-MLP groups.

Figure 2

MLPs affected mRNA expression in the intestines of HF and HF-MLP groups. (A) Volcano plots were used to characterize significantly different genes based on Log₂ (Fold change). (B) Heat map of transcriptome profile for HF and HF-MLP groups. Rows represent biological replicates and columns represent individual genes. Transcripts with higher expression levels in a sample are displayed in red and yellow, whereas those with lower expression levels are displayed in blue. (C) Enriched GO terms performed with Goatools package (https://github.com/tanghaibao/goatools) for DEGs between the HF and HF-MLP groups. (D) Enriched KEGG pathways performed with KOBAS 3.0 (http://bioinfo.org/kobas) for DEGs between HF and HF-MLP groups.

Figure 3

Integrating analysis of differentially modified m⁶A methylation and expressed genes in the intestines of HF and HF-MLP groups. (A) Four-quadrant graph representing the relationship between m⁶A methylation and gene expression. (B) GO analysis of genes with differential methylated m⁶A peaks and differential expression. (C) KEGG analysis of genes with differential methylated m⁶A peaks and differential expression. (D) Relative transcript levels of key genes (qRT-PCR validation, n = 8). ** indicates an extremely significant difference (p < 0.01; Independent sample t-test). (E) GSEA analysis for the lysosome and autophagy–animal pathway. (F) Peak abundance of m⁶A (IP) and expression (input) in nlrp1 and bnip3 in intestines of HF and HF-MLP groups. The dashed box represents the 3′UTR region of the gene. (G) Relative gene expression levels of methylase-related genes (n = 8). * indicates a significant difference (p < 0.05; Independent sample t-test); ** indicates an extremely significant difference (p < 0.01; Independent sample t-test).

Figure 4

The epigenetic regulatory mechanisms by which the inclusion of mulberry leaf polysaccharides (MLPs) in high-fat diet affect intestinal health in Megalobrama amblycephala. Upward arrows indicate significant up-regulation in mRNA level or m⁶A level, and downward arrows indicate significant down-regulation in mRNA level.