Comprehensive Analysis of Transcriptome-wide m6A Methylome Upon Clostridium perfringens Beta2 Toxin Exposure in Porcine Intestinal Epithelial Cells by m6A Sequencing

Abstract

Piglet diarrhea is a swine disease responsible for serious economic impacts in the pig industry. Clostridium perfringens beta2 toxin (CPB2), which is a major toxin of C. perfringens type C, may cause intestinal diseases in many domestic animals. N6-methyladenosine (m6A) RNA methylation plays critical roles in many immune and inflammatory diseases in livestock and other animals. However, the role of m6A methylation in porcine intestinal epithelial (IPEC-J2) cells exposed to CPB2 has not been studied. To address this issue, we treated IPEC-J2 cells with CPB2 toxin and then quantified methylation-related enzyme expression by RT-qPCR and assessed the m6A methylation status of the samples by colorimetric N6-methyladenosine quantification. The results showed that the methylation enzymes changed to varying degrees while the m6A methylation level increased (p < 0.01). On this basis, we performed N6-methyladenosine sequencing (m6A-seq) and RNA sequencing (RNA-seq) to examine the detailed m6A modifications and gene expression of the IPEC-J2 cells following CPB2 toxin exposure. Our results indicated that 1,448 m6A modification sites, including 437 up-regulated and 1,011 down-regulated, differed significantly between CPB2 toxin exposed cells and non-exposed cells (p < 0.05). KEGG pathway analysis results showed that m6A peaks up-regulated genes (n = 394) were mainly enriched in cancer, Cushing syndrome and Wnt signaling pathways, while m6A peaks down-regulated genes (n = 920) were mainly associated with apoptosis, small cell lung cancer, and the herpes simplex virus 1 infection signaling pathway. Furthermore, gene expression (RNA-seq data) analysis identified 1,636 differentially expressed genes (DEGs), of which 1,094 were up-regulated and 542 were down-regulated in the toxin exposed group compared with the control group. In addition, the down-regulated genes were involved in the Hippo and Wnt signaling pathways. Interestingly, the combined results of m6A-seq and RNA-seq identified genes with up-regulated m6A peaks but with down-regulated expression, here referred to as “hyper-down” genes (n = 18), which were mainly enriched in the Wnt signaling pathway. Therefore, we speculate that the genes in the Wnt signaling pathway may be modified by m6A methylation in CPB2-induced IPEC-J2 cells. These findings provide new insights enabling further exploration of the mechanisms underlying piglet diarrhea caused by CPB2 toxin.

m 6 A mRNA Methylation Analysis Provides Novel Insights into Pigmentation in Sheep Skin

“…As a result, the m 6 A peaks in black skin and white skin of sheep were mainly enriched in the CDS, 3‘UTR and 5‘UTR, especially in CDS region near the stop codon of the transcript. The result agrees with that in human, mouse, pig and sheep liver tissue [ 14 , 15 , 25 ]. The stability, localization, expression, and translation of mRNA were regulated by 3'UTR where multiple RNA-binding proteins bind to plays a regulatory role and protein interaction [ 27 ].…”

“…N6-methyladenosine (m 6 A), discovered in messenger RNA (mRNA) in 1974 from rat, is the most universal post-transcriptional modification of mRNA from bacteria, viruses, yeast, fruitflies, plants and mammals [ 8–12 ]. The m 6 A methylation is catalysed by methyltransferases (METTL3, METTL14, METTL16, CAPAM, METTL5, TRMT11, ZCCHC4, WTAP), removed by demethylases (FTO, ALKBH5) and recognized by reader proteins (YTHDF1/2/3,) [ 13 , 14 ]. M 6 A modifications, regulating the stability, splicing, translation, and degradation of mRNAs, may play important roles in growth, reproduction, nerve development, fat metabolism, immune responses, tumour invasion and other physiological processes [ 15–17 ].…”

Comprehensive analysis of pre-mRNA alternative splicing regulated by m6A methylation in pig oxidative and glycolytic skeletal muscles

“…We suspected that RNA binding proteins (RBPs) enriched in the neighbor region of skipping exon may participate in the regulation of SE with m6A. We expectedly found a consensus motif RRACH (R: A/ G, H: A/C/U) that is the typical m6A motif conserved in mammals [ 39 – 41 ], indicating the high accuracy in calling m6A peak (Fig. 4 F).…”

Comprehensive analysis of LncRNAs modified by m6A methylation in sheep skin

“…universal mRNA modification present across various species [22][23][24][25]. The m 6 A methylation is recognized, catalyzed and removed by reader proteins, methyltransferases and demethylases, respectively [26,27]. M 6 A modifications may play important roles in metabolism, growth, development, immune responses, reproduction, and other physiological processes [28][29][30].…”