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The role of N6-methyladenosine RNA methylation in the heat stress response of sheep (Ovis aries)

  • Zengkui Lu
  • Youji Ma
  • Qing Li
  • Enmin Liu
  • Meilin Jin
  • Liping ZhangEmail author
  • Caihong WeiEmail author
Original Paper
  • 61 Downloads

Abstract

With the intensive development of the sheep industry and increasing global temperatures, heat stress in sheep has become an increasingly severe and important issue in recent years. The level of N6-methyladenosine (m6A) RNA methylation changes in response to stress plays important roles in stress responses. However, the role of m6A in the heat stress response of sheep remains unclear. To explore this issue, we measured heat stress protein (HSP) expression, liver function indexes, m6A on RNA, m6A-related enzyme expression, and tissue damage in sheep that had been subjected to heat stress. At the transcriptome level, our results showed significant increases in m6A on RNA and increased mRNA levels of HSPs (HSP70, HSP90, and HSP110) and m6A-related enzymes [METTL3 (methyltransferase-like 3), METTL14 (methyltransferase-like 14), WTAP (wilms tumor 1-associated protein), FTO (fat mass and obesity-associated protein), ALKBH5 (alkB homologue 5), YTHDF1-3 (YTH domain family proteins), and YTHDC1-2 (YTH domain-containing proteins)] following heat stress. At the protein level, the expression of METTL3, YTHDF1-2, and YTHDC2 showed no significant differences following heat stress. However, in contrast to its mRNA level after heat stress, the protein expression of YTHDF3 was reduced, while the expression of HSPs (HSP70, HSP90, and HSP110), METTL14, WTAP, FTO, ALKBH5, YTHDF3, and YTHDC1 increased in line with their measured mRNA levels. Histological experiments revealed that heat stress caused varying degrees of damage to sheep liver tissue. Moreover, immunohistochemical staining indicated that the m6A-related enzymes were expressed in sheep hepatocytes, and differences in expression patterns were observed between the control and heat stress groups. In summary, differences in the level of m6A and the expression of m6A-related enzymes in the liver of sheep were observed after heat stress. This indicates that m6A is involved in the regulation of heat stress in sheep. Our findings provide a new avenue for studying the responses to heat stress in sheep.

Keywords

Heat stress Hu sheep N6-methyladenosine RNA methylation m6A-related enzymes 

Notes

Author contributions

Caihong Wei, Liping Zhang, and Zengkui Lu conceived and designed the experiments; Zengkui Lu, Youji Ma, Enmin Liu, Qing Li, and Meilin Jin collected samples; and Zengkui Lu performed the experiments and wrote the paper.

Funding information

This work was supported by the National Modern Agricultural Industry Technology Fund for Scientists in Sheep Industry System (No. CARS-38) and the National Natural Science Foundation of China (No.31672380).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

12192_2018_965_Fig7_ESM.png (742 kb)
Supplementary Fig. S1

qRT-PCR standard curves. Amplification efficiencies of all primers range between 95% and 105%, which meets the experimental requirements of qRT-PCR. (PNG 742 kb)

12192_2018_965_MOESM1_ESM.tiff (314 kb)
High Resolution Image (TIFF 314 kb)
12192_2018_965_Fig8_ESM.png (1.2 mb)
Supplementary Fig. S2

qRT-PCR amplification curves. Amplification curves of all primers are smooth, without amplification in the beginning. The starting moment of peak is normal. (PNG 1277 kb)

12192_2018_965_MOESM2_ESM.tiff (464 kb)
High Resolution Image (TIFF 463 kb)
12192_2018_965_Fig9_ESM.png (939 kb)
Supplementary Fig. S3

qRT-PCR melting curves. Melting curves of all primers present a single narrow peak, which indicates the absence of redundant products and primer dimers. (PNG 939 kb)

12192_2018_965_MOESM3_ESM.tiff (693 kb)
High Resolution Image (TIFF 693 kb)
12192_2018_965_MOESM4_ESM.docx (32 kb)
Supplementary Table S1 m6A-related enzymes and their roles in RNA metabolism. (DOCX 31 kb)
12192_2018_965_MOESM5_ESM.docx (23 kb)
Supplementary Table S2 The primers used in this study for qRT-PCR. (DOCX 22 kb)
12192_2018_965_MOESM6_ESM.docx (20 kb)
Supplementary Table S3 The antibodies used in this study for western blotting and immunohistochemistry. (DOCX 20 kb)

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Copyright information

© Cell Stress Society International 2019

Authors and Affiliations

  1. 1.College of Animal Science and TechnologyGansu Agricultural UniversityLanzhouChina
  2. 2.Key Laboratory of Animal Genetics and Breeding and Reproduction of Ministry of Agriculture, Institute of Animal ScienceChinese Academy of Agricultural SciencesBeijingChina

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