The effect of heat stress on frame switch splicing of X-box binding protein 1 gene in horse

Lee, Hyo Gun;Khummuang, Saichit;Youn, Hyun-Hee;Park, Jeong-Woong;Choi, Jae-Young;Shin, Teak-Soon;Cho, Seong-Keun;Kim, Byeong-Woo;Seo, Jakyeom;Kim, Myunghoo;Park, Tae Sub;Cho, Byung-Wook

  • Received : 2018.10.11
  • Accepted : 2018.12.15
  • Published : 2019.08.01


Objective: Among stress responses, the unfolded protein response (UPR) is a well-known mechanism related to endoplasmic reticulum (ER) stress. ER stress is induced by a variety of external and environmental factors such as starvation, ischemia, hypoxia, oxidative stress, and heat stress. Inositol requiring enzyme $1{\alpha}$ ($IRE1{\alpha}$)-X-box protein 1 (XBP1) is the most conserved pathway involved in the UPR and is the main component that mediates $IRE1{\alpha}$ signalling to downstream ER-associated degradation (ERAD)- or UPR-related genes. XBP1 is a transcription factor synthesised via a novel mechanism called 'frame switch splicing', and this process has not yet been studied in the horse XBP1 gene. Therefore, the aim of this study was to confirm the frame switch splicing of horse XBP1 and characterise its dynamics using Thoroughbred muscle cells exposed to heat stress. Methods: Primary horse muscle cells were used to investigate heat stress-induced frame switch splicing of horse XBP1. Frame switch splicing was confirmed by sequencing analysis. XBP1 amino acid sequences and promoter sequences of various species were aligned to confirm the sequence homology and to find conserved cis-acting elements, respectively. The expression of the potential XBP1 downstream genes were analysed by quantitative real-time polymerase chain reaction. Results: We confirmed that splicing of horse XBP1 mRNA was affected by the duration of thermal stress. Twenty-six nucleotides in the mRNA of XBP1 were deleted after heat stress. The protein sequence and the cis-regulatory elements on the promoter of horse XBP1 are highly conserved among the mammals. Induction of putative downstream genes of horse XBP1 was dependent on the duration of heat stress. We confirmed that both the mechanisms of XBP1 frame switch splicing and various binding elements found in downstream gene promoters are highly evolutionarily conserved. Conclusion: The frame switch splicing of horse XBP1 and its dynamics were highly conserved among species. These results facilitate studies of ER-stress in horse.


Thoroughbred;Heat Stress;X-box Binding Protein 1;Quantitative Real-time Polymerase Chain Reaction (qRT-PCR)


  1. Wu J, Kaufman RJ. From acute ER stress to physiological roles of the unfolded protein response. Cell Death Differ 2006;13:374-84.
  2. Calfon M, Zeng H, Urano F, et al. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 2002;415:92-6.
  3. Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 2001;107:881-91.
  4. Uemura A, Oku M, Mori K, Yoshida H. Unconventional splicing of XBP1 mRNA occurs in the cytoplasm during the mammalian unfolded protein response. J Cell Sci 2009;122:2877-86.
  5. Mori K. Frame switch splicing and regulated intramembrane proteolysis: key words to understand the unfolded protein response. Traffic 2003;4:519-28.
  6. Roy B, Lee AS. The mammalian endoplasmic reticulum stress response element consists of an evolutionarily conserved tripartite structure and interacts with a novel stress-inducible complex. Nucleic Acids Res 1999;27:1437-43.
  7. Yoshida H, Haze K, Yanagi H, Yura T, Mori K. Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins. Involvement of basic leucine zipper transcription factors. J Biol Chem 1998;273:33741-9.
  8. Yoshida H, Okada T, Haze K, et al. ATF6 activated by proteolysis binds in the presence of NF-Y (CBF) directly to the cisacting element responsible for the mammalian unfolded protein response. Mol Cell Biol 2000;20:6755-67.
  9. Marlin DJ, Scott CM, Roberts CA, Casas I, Holah G, Schroter RC. Post exercise changes in compartmental body temperature accompanying intermittent cold water cooling in the hyperthermic horse. Equine Vet J 1998;30:28-34.
  10. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25:402-8.
  11. Kearse M, Moir R, Wilson A, et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 2012;28:1647-9.
  12. Xu X, Gupta S, Hu W, McGrath BC, Cavener DR. Hyperthermia induces the ER stress pathway. PLoS One 2011;6:e23740.
  13. Kokame K, Kato H, Miyata T. Identification of ERSE-II, a new cis-acting element responsible for the ATF6-dependent mammalian unfolded protein response. J Biol Chem 2001;276:9199-205.
  14. Kokame K, Agarwala KL, Kato H, Miyata T. Herp, a new ubiquitin-like membrane protein induced by endoplasmic reticulum stress. J Biol Chem 2000;275:32846-53.
  15. Yamamoto K, Yoshida H, Kokame K, Kaufman RJ, Mori K. Differential contributions of ATF6 and XBP1 to the activation of endoplasmic reticulum stress-responsive cis-acting elements ERSE, UPRE and ERSE-II. J Biochem 2004;136:343-50.
  16. Yoshida H, Matsui T, Hosokawa N, Kaufman RJ, Nagata K, Mori K. A time-dependent phase shift in the mammalian unfolded protein response. Dev Cell 2003;4:265-71.
  17. Yamamoto K, Suzuki N, Wada T, et al. Human HRD1 promoter carries a functional unfolded protein response element to which XBP1 but not ATF6 directly binds. J Biochem 2008;144:477-86.
  18. Kaneko M, Ishiguro M, Niinuma Y, Uesugi M, Nomura Y. Human HRD1 protects against ER stress-induced apoptosis through ER-associated degradation. FEBS Lett 2002;532:147-52.
  19. Damiano F, Tocci R, Gnoni GV, Siculella L. Expression of citrate carrier gene is activated by ER stress effectors XBP1 and ATF6alpha, binding to an UPRE in its promoter. Biochim Biophys Acta Gene Regul Mech 2015;1849:23-31.
  20. Hoozemans JJ, Veerhuis R, Van Haastert ES, et al. The unfolded protein response is activated in Alzheimer's disease. Acta Neuropathol 2005;110:165-72.
  21. Ozcan U, Cao Q, Yilmaz E, et al. Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes. Science 2004;306:457-61.
  22. Dong D, Ni M, Li J, et al. Critical role of the stress chaperone GRP78/BiP in tumor proliferation, survival, and tumor angiogenesis in transgene-induced mammary tumor development. Cancer Res 2008;68:498-505.
  23. Yamasaki S, Yagishita N, Tsuchimochi K, Nishioka K, Nakajima T. Rheumatoid arthritis as a hyper-endoplasmic-reticulumassociated degradation disease. Arthritis Res Ther 2005;7:181.
  24. Mayer MP, Bukau B. Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol Life Sci 2005;62:670.
  25. Lee K, Tirasophon W, Shen X, et al. IRE1-mediated unconventional mRNA splicing and S2P-mediated ATF6 cleavage merge to regulate XBP1 in signaling the unfolded protein response. Genes Dev 2002;16:452-66.
  26. Wang Y, Shen J, Arenzana N, Tirasophon W, Kaufman RJ, Prywes R. Activation of ATF6 and an ATF6 DNA binding site by the endoplasmic reticulum stress response. J Biol Chem 2000;275:27013-20.
  27. Cox JS, Walter P. A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response. Cell 1996;87:391-404.
  28. Mori K, Ogawa N, Kawahara T, Yanagi H, Yura T. Palindrome with spacer of one nucleotide is characteristic of the cis-acting unfolded protein response element in Saccharomyces cerevisiae. J Biol Chem 1998;273:9912-20.


Supported by : Rural Development Administration, National Research Foundation of Korea (NRF)