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Asian-Australas J Anim Sci > Accepted Articles
DOI: https://doi.org/10.5713/ajas.18.0757    [Accepted] Published online January 4, 2019.
The effect of heat stress on frame switch splicing of X-box binding protein 1 gene in horse
Hyo Gun Lee1, Saichit Khummuang1, Hyun-Hee Youn1, Jeong-Woong Park1, Jae-Young Choi1, Teak-Soon Shin1, Seong-Keun Cho1, Byeong-Woo Kim1, Jakyeom Seo1, Myunghoo Kim1, Tae Sub Park2, Byung-wook Cho1,* 
1Pusan National University, Miryang, Korea
2Seoul National University, Pyeongchang , Korea
Correspondence:  Byung-wook Cho, Tel: 821092079392, Fax: 82553505519, Email: bwcho@pusan.ac.kr
Received: 11 October 2018   • Revised: 26 November 2018   • Accepted: 15 December 2018
Abstract
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 1α (IRE1α)-X-box protein 1 (XBP1) is the most conserved pathway involved in the UPR and is the main component that mediates IRE1α 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 qRT-PCR.

Results

We confirmed that splicing of horse XBP1 mRNA was affected by the duration of thermal stress. 26 nucleotides in the exon 4 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.
Keywords: Thoroughbred; Heat Stress; X-box Binding Protein 1; qRT-PCR


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