Identification of Yak’s TLR4 Alternative Spliceosomes and Bioinformatic Analysis of TLR4 Protein Structure and Function

SIMPLE SUMMARY: In this study, yak’s TLR4 gene alternative spliceosomes were investigated using PCR amplification and cloning with an aim to improve disease-resistance in yaks and promote efficient utilization of yak’s resources. qRT-PCR was used to evaluate the expression levels of two alternatively spliced TLR4 transcripts in seven distinct yak tissues. To predict the function of proteins expressed by each TLR4 spliceosome, TLR4 protein structure and function were analyzed bioinformatically. Besides, two alternative spliceosomes of yak’s TLR4 gene were also identified, which were in line with predicted variants of the TLR4 gene in NCBI. These two alternative spliceosomes of the TLR4 gene were expressed in each tissue; however, the expression levels of these spliceosomes were significantly different in different tissue. We also observed that deletion of exon-2 in TLR4 affected the function of the corresponding protein. This study will lay a theoretical foundation for future studies on the role of two variants of yak’s TLR4 gene in disease resistance. Besides, data from this study could be analyzed further to explore the molecular mechanism associated with disease-resistance in the yak. ABSTRACT: In this study, the yak’s TLR4 gene alternative spliceosomes were investigated using PCR amplification and cloning to improve disease-resistance in yak and promote efficient utilization of yak’s resources. qRT-PCR was used to determine the expression levels of two alternatively spliced transcripts of the TLR4 gene in seven distinct tissues. To predict the function of proteins expressed by each TLR4 spliceosome, bioinformatic analysis of yak’s TLR4 protein structure and function was performed, which led to the identification of two alternative spliceosomes of yak’s TLR4 gene. The TLR4-X1 sequence length was 2526 bp, and it encoded full-length TLR4 protein (841 amino acids). The sequence length of the exon-2 deleted TLR4-X2 sequence was 1926 bp, and it encoded truncated TLR4 protein (641 amino acids). TLR4-X2 sequence was consistent with the predicted sequence of the TLR4 gene in GenBank. Each tissue showed significantly different expression levels of these two alternative spliceosomes. As per the bioinformatic analysis of the structure and function of TLR4 protein, deletion of exon-2 in the TLR4 gene resulted in frameshift mutations of the reading frame in the corresponding protein, which altered its ligand-binding and active sites. Besides, biological property such as substrate specificity of truncated TLR4 protein was also altered, leading to altered protein function. This study has laid a theoretical foundation for exploring the role of two variants of the TLR4 gene in yak’s disease resistance. Besides, this study’s data could be analyzed further to explore the molecular mechanism associated with disease-resistance in the yak.