The role of RNA epigenetic modification-related genes in the immune response of cattle to mastitis induced by Staphylococcus aureus

Abstract

Objective: RNA epigenetic modifications play an important role in regulating immune response of mammals. Bovine mastitis induced by Staphylococcus aureus (S. aureus) is a threat to the health of dairy cattle. There are numerous RNA modifications, and how these modification-associated enzymes systematically coordinate their immunomodulatory effects during bovine mastitis is not well reported. Therefore, the role of common RNA modificationrelated genes (RMRGs) in bovine S. aureus mastitis was investigated in this study.

Methods: In total, 80 RMRGs were selected for this study. Four public RNA-seq data sets about bovine S. aureus mastitis were collected and one additional RNA-seq data set was generated by this study. Firstly, quantitative trait locus (QTL) database, transcriptome-wide association studies (TWAS) database and differential expression analyses were employed to characterize the potential functions of selected enzyme genes in bovine S. aureus mastitis. Correlation analysis and weighted gene co-expression network analysis (WGCNA) were used to further investigate the relationships of RMRGs from different types at the mRNA expression level. Interference experiments targeting the m6A demethylase FTO and utilizing public MeRIP-seq dataset from bovine Mac-T cells were used to investigate the potential interaction mechanisms among various RNA modifications.

Results: Bovine QTL and TWAS database in cattle revealed associations between RMRGs and immune-related complex traits. S. aureus challenged and control groups were effectively distinguished by principal component analysis based on the expression of selected RMRGs. WGCNA and correlation analysis identified modules grouping different RMRGs, with highly correlated mRNA expression. The m6A modification gene FTO showed significant effects on the expression of m6A and other RMRGs (such as NSUN2, CPSF2, and METTLE), indicating complex co-expression relationships among different RNA modifications in the regulation of bovine S. aureus mastitis.

Conclusion: RNA epigenetic modification genes play important immunoregulatory roles in bovine S. aureus mastitis, and there are extensive interactions of mRNA expression among different RMRGs. It is necessary to investigate the interactions between RNA modification genes regulating complex traits in the future.

Keywords: Mastitis; RNA Modification Gene; RNA-seq; Staphylococcus aureus.

Figure 1

Technical route of this study.

Figure 2

The top 20 immunity-associated quantitative trait loci (QTLs) identified by RNA modification-related genes.

Figure 3

Principal component analysis (PCA) and Log₂fold change (FC) for gene expression of RNA modification-related genes in control and Staphylococcus aureus challenged groups. (A) presents the PCA results of control and S. aureus challenged groups from all datasets. (B) presents Log₂FC between the S. aureus challenged group and control group in each data. * Means the p-value of less than 0.05. (C)-(D) Presents the normalized expression levels of the NSUN2 and YTHDF2 genes across eight distinct groups. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Paired t-test representing the significantly consistent trend group of gene changes.

Figure 4

RMRGs exhibit interactions and participate in immune regulation. (A) Weighted correlation network analysis for different Staphylococcus aureus challenged groups. (B) Correlation of gene expression among RMRGs within Module 8. (C)–(D) Gene ontology analysis of the genes for modules 8 and 14. RMRGs, RNA modification-related genes. * p<0.05, ** p<0.01, and *** p<0.001, respectively.

Figure 5

FTO influence the expression of RNA-modified genes containing m⁶A modification sites. (A) Interference result for FTO. **** p<0.001. (B)–(C) Total cell extracts were harvested from siFTO and NC Mac-T cells and subjected to Western blot. (D) Expression changes of CPSF2 gene after cellular-level interference with FTO. * p<0.05. (E) CPSF2 gene expression significantly correlated with FTO. (F) Variations in the m⁶A modification peak within the CPSF2 gene between the control group and Staphylococcus aureus challenged group. (G) Kyoto encyclopedia of genes and genomes analysis of differentially expressed genes (DEGs) with m⁶A peaks post-FTO interference. Circle attributes indicate p-value significance and gene count. (H) Gene ontology enrichment of DEGs with m⁶A peaks post-FTO interference. Circle attributes indicate p-value significance and gene count. m⁶A, N6-methyladenosine.