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. 2022 Dec 14;23(1):829.
doi: 10.1186/s12864-022-09067-6.

The alteration of N6-methyladenosine (m6A) modification at the transcriptome-wide level in response of heat stress in bovine mammary epithelial cells

Ying Qi  1 , Yiming Zhang  1 , Jing Zhang  1 , Jing Wang  1 , Qiuling Li  2
Affiliations

The alteration of N6-methyladenosine (m6A) modification at the transcriptome-wide level in response of heat stress in bovine mammary epithelial cells

Ying Qi et al. BMC Genomics. .

Abstract

Background: Heat stress has a substantial negative economic impact on the dairy industry. N6-methyladenosine (m6A) is the most common internal RNA modification in eukaryotes and plays a key role in regulating heat stress response in animals. In dairy cows, however, this modification remains largely unexplored. Therefore, we examined the effects of heat stress on the m6A modification and gene expression in bovine mammary epithelial cells to elucidate the mechanism of heat stress response. In this study, Mammary alveolar cells-large T antigen (MAC-T) cells were incubated at 37 °C (non-heat stress group, NH) and 40 °C (heat stress group, H) for 2 hours, respectively. HSP70, HSF1, BAX and CASP3 were up regulated in H group compared with those in the NH group.

Results: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were conducted to identify m6A peaks and to produce gene expression data of MAC-T cells in the H and NH groups. In total, we identified 17,927 m6A peaks within 9355 genes in the H group, and 18,974 peaks within 9660 genes in the NH groups using MeRIP-seq. Compared with the NH group, 3005 significantly differentially enriched m6A peaks were identified, among which 1131 were up-regulated and 1874 were down-regulated. In addition, 1502 significantly differentially expressed genes were identified using RNA-seq, among which 796 were up-regulated and 706 were down-regulated in the H group compared to the NH group. Furthermore, 199 differentially expressed and synchronously differentially methylated genes were identified by conjoint analysis of the MeRIP-seq and RNA-seq data, which were subsequently divided into four groups: 47 hyper-up, 53 hyper-down, 59 hypo-up and 40 hypo-down genes. In addition, GO enrichment and KEGG analyses were used to analyzed the potential functions of the genes in each section.

Conclusion: The comparisons of m6A modification patterns and conjoint analyses of m6A modification and gene expression profiles suggest that m6A modification plays a critical role in the heat stress response by regulating gene expression.

Keywords: Heat stress; MAC-T; MeRIP-seq; N6-methyladenosine; m6A.

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Conflict of interest statement

All the authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Effect of heat stress on HSP70, HSF1, BAX, and CASP3 expression in MAC-T cells. Expression of HSP70 (a), HSF1 (b), BAX (c), and CASP3 (d) was detected through qRT-PCR
Fig. 2
Fig. 2
m6A topological patterns in MAC-T cells. a Metagene plots showing the m6A peak density distribution across the transcripts in the NH and H groups. Gene segments distribution of m6A peaks in the H (b) and NH (c) groups. The top sequence motifs enriched within m6A peaks in the H (d) and NH (e) groups. (f) Proportion of genes containing different numbers of m6A peaks in the NH and H groups
Fig. 3
Fig. 3
Differential m6A peaks between H and NH groups. a Venn diagram showing the overlap of m6A peaks between H and NH groups. b Venn diagram showing the overlap of m6A containing genes between H and NH groups. c Volcano plots of the significantly different m6A peaks. d GO annotation of differential m6A peak-related genes. e KEGG enrichment analysis of differential m6A peaks related genes
Fig. 4
Fig. 4
Conjoint analysis of MeRIP-seq and RNA-seq data. a Venn diagram showing the numbers of genes expressed in the H and NH groups. b Volcano plots showing differentially expressed genes. c Four-quadrant diagram showing the distribution of genes with significant changes in both m6A modification and mRNA expression. d GO annotation of genes with significant changes in m6A modification and mRNA expression. e KEGG enrichment analysis of genes with significant changes in m6A modification and mRNA expression
Fig. 5
Fig. 5
The distribution of m6A peaks in HSP mRNA. Visualization of HSPA1A, HSPA5, HSPA8, and HSPH1 using Integrative Genomics Viewer (IGV) software. The blue box indicates an exon and the blue line indicates an intron
Fig. 6
Fig. 6
Effect of heat stress on cells through the m6A modification. The speculated molecular mechanism by which heat stress affects m6A is shown. The cell signaling pathways enriched in the KEGG analysis are indicated
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References

    1. Wei CM, Gershowitz A, Moss B. Methylated nucleotides block 5′ terminus of HeLa cell messenger RNA. Cell. 1975;4(4):379–386. doi: 10.1016/0092-8674(75)90158-0. - DOI - PubMed
    1. Boccaletto P, Machnicka MA, Purta E, Piatkowski P, Baginski B, Wirecki TK, et al. MODOMICS: a database of RNA modification pathways. 2017 update. Nucleic Acids Res. 2018;46(D1):D303–D307. doi: 10.1093/nar/gkx1030. - DOI - PMC - PubMed
    1. Frye M, Harada BT, Behm M, He C. RNA modifications modulate gene expression during development. Science. 2018;361(6409):1346–1349. doi: 10.1126/science.aau1646. - DOI - PMC - PubMed
    1. Cantara WA, Crain PF, Rozenski J, McCloskey JA, Harris KA, Zhang X, et al. The RNA modification database, RNAMDB: 2011 update. Nucleic Acids Res. 2011;39(Database issue):D195–D201. doi: 10.1093/nar/gkq1028. - DOI - PMC - PubMed
    1. Clancy MJ, Shambaugh ME, Timpte CS, Bokar JA. Induction of sporulation in Saccharomyces cerevisiae leads to the formation of N6-methyladenosine in mRNA: a potential mechanism for the activity of the IME4 gene. Nucleic Acids Res. 2002;30(20):4509–4518. doi: 10.1093/nar/gkf573. - DOI - PMC - PubMed