1. Introduction
Yaks are long-haired bovine species and domesticated cattle that are found at plateau located between 3000–5000 meters above sea level. Native to China, 95% of the world’s yak population is distributed around the Qinghai-Tibet plateau in China [
1]. Apart from meat and milk, yaks are widely used for labor. Besides, yaks provide livelihood and multiple useful resources, such as hair and skin, for the Qinghai-Tibet plateau’s pastoral areas. Thus, yak is also known as “boat on the plateau” and “omnipotent livestock [
2].” However, yaks have a low survival rate due to low immunity [
3]. Recently, due to the rising standard of living, the yak market has gradually expanded. Thus, improving the yak’s immunity is crucial for expanding the yak market.
Toll-like receptors (TLRs) are pathogenic pattern recognition receptors (PRPs), which were discovered in 1997 [
4,
5]. TLRs mediate both innate and acquired immunity and play a crucial role in the immune response [
6]. Till now, a total of 13 TLRs have been identified in mammals [
7]. TLR4 is one of the earliest discovered and a highly characterized member of the TLR family [
8]. It is mainly distributed on the membrane of monocytes, macrophages, NK cells, and adipocytes [
9]. TLR4 recognizes highly conserved pathogen-associated molecular patterns (PAMPs) receptors, which are expressed on pathogenic microorganisms [
10]. TLR4–PAMP interaction activates intracellular signaling in the early stages of pathogenic invasion to induce the expression crucial immunogenic genes [
11]. At the same time, immunogenic genes promote cell synthesis and secretion of cytokines like IFN, IL and TNF. These cytokines transmit upstream signals to downstream and stimulate the natural immune response to kill and elim-inate pathogens, which could further activate the adaptive immune response [
12]. A recent study has shown that
TLR4 gene knockout in mice resulted in disorders of visceral adipose tissue and immune cells [
13] and that TLR4 was involved in crucial regulatory roles of the innate immune system. Multiple studies have explored the
TLR4 gene in domestic animals [
14,
15] and poultry [
16], but the
TLR4 gene in yak remains unexplored. Unraveling the expression and function of the yak’s
TLR4 gene alternative spliceosomes will be highly significant for future immunogenic studies. This study provides a theoretical basis for exploring the yak’s innate immune system; besides, the data could direct the breeding of high-quality disease-resistant yaks.
Alternative splicing (AS) is the process where different mature mRNA spliced isoforms are produced from a single pre-mRNA through different splicing methods and splice sites [
10]. It is a crucial process that regulates gene expression and proteomal diversity [
17]. Alternative splicing was first discovered in adenoviruses [
18] and subsequently, in mammals [
19]. As per the previous studies, alternative splicing plays a crucial role in the generation of receptor diversity, regulation of growth and development, specifically the nervous and immune system; besides, it exerts functional diversity and response sensitivity to each tissue [
20]. Multiple pathogenic mutations are co-related to the alternative splicing of genes. Mutations in the conserved sequence of splicing sites, i.e., cis- and trans-acting elements, post alternative splicing, resulted in the physiological abnormalities [
21]. Deciphering the correlation between the regulatory mechanism of alternative spliceosomes and diseases could unravel the pathogenic mechanism and facilitate the development of therapeutic agents for multiple immunogenic disorders.
In this study, the CDS region of the yak’s
TLR4 gene was amplified using primers, which were designed using yak’s
TLR4 gene’ predicted sequences (XM_005891938.1 and XM_014477047.1), obtained using NCBI (
https://www.ncbi.nlm.nih.gov/). Apart from identifying alternative spliceosomes, novel alternative spliceosomes of the
TLR4 gene were also explored. qRT-PCR was employed to detect the expression levels of two alternative spliceosomes of the
TLR4 gene in distinct tissues. Furthermore, functional bioinformatics analysis was performed for each spliceosome. This study aimed to reveal the structure, expression characteristics, and functions of each yak’s
TLR4 gene spliceosome, along with structural and functional differences between TLR4 full-length and truncated protein from exon-2 deleted
TLR4 gene. In this study, we investigated the effect of the
TLR4 gene’s alternative splicing on the function of the corresponding TLR4 protein. This study provides primary data for future research on yak’s innate immunity.
2. Materials and Methods
2.1. Experimental Animals
All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee of the Lanzhou Institute of Husbandry and Pharmaceutical Science of the Chinese Academy of Agricultural Sciences (Approval No: NKMYD201904; dated: 26 October 2019). In this experiment, three healthy male yaks were procured from Datong Yak Factory, located in Qinghai Province, China. All the three yaks were euthanized, and their heart, liver, spleen, lung, kidney, back muscles, and the subcutaneous adipose tissue of the back were harvested and immediately transported to the Lanzhou Institute of Husbandry and Pharmaceutical Sciences Chinese Academy of Agricultural Sciences, Gansu Province, China on dry ice for further experiments.
2.2. Sample Preparation and cDNA Synthesis
Around 3 g of the tissue samples from each tissue (heart, liver, spleen, lung, kidney, muscles, and fat tissue) were used for RNA extraction. Total RNA was extracted from each tissue using TRIzol reagent (Takara Bio Inc., Dalian, China). To synthesize cDNA from RNA, each RNA sample was diluted to 500 ng/μL and reverse transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Takara Bio Inc., Dalian, China). The synthesized cDNA was stored at −80 °C until further use. The RNA concentration and OD260/280 ratio of the samples were determined using NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). RNA concentration and OD260/280 ratio of the samples ranged between 500–5000 ng/μL and 1.9–2.1, respectively. The RNA extraction metrics and tissue-specific metadata were showed in
Table S1. Ribonucleic acid quality was assessed by evaluating the 28S and the 18S rRNA bands on a 1% agarose electrophoretic gel.
2.3. Primer Design and Synthesis
Based on two predicted alternative spliceosome sequences of the
TLR4 gene, obtained using NCBI (
https://www.ncbi.nlm.nih.gov/) (XM_00589193.1 and XM_014477041.1), three pairs of primers (P1, P2, P3) were designed using Primer Premier 5.0 software [
22] to amplify yak’s
TLR4 gene’s CDS region (
Table 1) for the identification of novel spliceosomes and two predicted spliceosomes as mentioned in NCBI. Xi’an Qingke Biotechnology Co., Ltd (Xi’an, China) synthesized the primers.
2.4. TLR4 Variants, PCR Amplification, Cloning, and Sequencing
PCR reaction mixture contained 1 μL of forward primer and 1 μL of reverse primer, 12.5 μL of 2 × Taq Master Mix, 1 μL of cDNA from yak’s spleen tissue, and 9.5 μL of ddH2O. Initial denaturation was carried out at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 58 °C for 50 s, extension at 72 °C for 3 min, and a final extension at 72 °C for 10 min. PCR products were detected using 1.2% agar-gel electrophoresis (AGE), visualized, and stored in the automatic digital gel imaging system.
The PCR products were recovered from the agarose-gel using the EasyPure Quick Gel Extraction Kit (Quanshijin Biotechnology Co., Ltd., Beijing, China), as per the manufacturer’s instructions. To prepare recombinant plasmids, pMD19-T vectors (Takara Bio Inc., Dalian, China) were mixed with PCR products and placed in a water bath at 16 °C overnight. These recombinant plasmids were incubated with BL21 competent cells (Takara Bio Inc., Dalian, China) to transform the recombinant plasmids into the BL21 competent cells. 200 μL of bacterial suspension was spread on LB agar (Amp+) plates and incubated at 37 °C for 12 h. Later, single colonies were selected and inoculated into the 5 mL LB broth (Amp+) and incubated with constant shaking at 37 °C for 12 h. Post-incubation, positive bacterial clones were validated using PCR amplification and sequenced by Xi’an Gt Biotechnology Co., Ltd. (Xi’an, China).
2.5. Bioinformatics Analysis of TLR4 Protein Structure and Function
The biological function of a protein can be attributed to its 3D-structure [
23]. However, the preparation of protein crystals for analyzing the 3D-structure of proteins is a tedious task. In this study, I-TASSER [
24] (
http://zhanglab.ccmb.med.umich.edu/I-TASSER/), a prediction software, was employed to predict the structure and function of the protein and to construct the 3D-model of the full-length TLR4 and truncated TLR4 protein, encoded by exon-2 deleted
TLR4 gene. Starting from the amino acid sequence, I-TASSER first uses LOMETS [
25] (a multi-threading algorithm consisting of several separate threaded programs) to identify homologous structure templates or super secondary structure segments from the PDB library [
26] (
http://www.pdo.org/pdb/hoine/home.do). To construct the complete structure, it reassembles the LOMETS template, excises continuous aligned fragment structure from the super secondary structure segment, precisely-optimizes the structure model, and determines the free energy conformation. To deduce the biological function of the target protein, the I-TASSER model was matched with the protein obtained from the BioLip library [
27], and its functions, ligand binding site, enzyme classification, and other characteristics were inferred from the BioLip template. A phylogenetic tree was constructed based on the amino acid sequences of two alternative spliceosomes of TLR4, as described previously by Xiaoyan Zhang [
28].
2.6. Quantitative Real-Time PCR (qRT-PCR) Based Analysis of TLR4-X1 and TLR4-X2 mRNA Expression
Fluorescence quantitative primers (P4, P5) were designed based on the sequences of two alternative
TLR4 spliceosomes obtained by cloning (
Table 1). The glyceraldehyde 3-phosphate dehydrogenase gene (
GAPDH) was used as the reference gene. Semi-quantitative RT-PCR was used to determine the expression levels of
TLR4 transcript in all the yak tissue samples. Fluorescent quantitative PCR reaction system and reaction conditions were as described previously by Wangsheng Zhao [
29], and the relative expression of genes was estimated using the 2
−ΔΔCT [
30] method. Each reaction was repeated three times to obtain the Ct value of each sample. Statistical differences between the expression levels of the
TLR4 splice variants were determined through ANOVA. SPSS version 21.0 [
31] was used to perform statistical analyses.
p < 0.05 or
p < 0.01 was considered as statistically significant [
32].
4. Discussion
Alternative splicing is a widely occurring phenomenon and closely related to altered protein functions, such as altered amino or carboxy terminus of a protein or addition/deletion of the functional region [
37]. Till now, seven alternative splicing methods [
38]: variable 3′ splice sites, variable 5′ splice sites, intron retention, mutual exclusion exons, variable initial exon, variable terminal exon, have been proposed. For the first time, in this study, the entire exon-2 was deleted from the
TLR4 transcript to produce TLR4-X2 mRNA. Previous studies have reported two alternatively spliced variants of the
TLR4 gene in duck [
10] and mice [
39]; three alternatively spliced variants in sheep [
10] and pig [
40], and four alternatively spliced variants in human [
39]. However, alternatively spliced variant of the yak’s
TLR4 gene have not been reported so far.
In this study, to identify the alternatively spliced variants of yak’s
TLR4 gene, cDNA from yak’s spleen was amplified using PCR. PCR products were visualized on agarose gel electrophoresis. Three bands appeared in one lane (
Figure 1a); these bands were recovered from agarose-gel and later cloned. Two bands (929 bp and 750 bp) were consistent with the predicted sequence of alternative spliceosome obtained using NCBI. These three sequences were aligned, and it was found that the starting sequence of the sample in the third lane (300 bp) was identical to the primer’s sequence. Since this sequence might have been mistaken for the upstream primer by the system and later eliminated from the synthesized sequence, the pair of P2 primers (P2) was used to assess the third band (300 bp) on agarose gel electrophoresis. We observed that one lane contained only two bands (756 bp and 600 bp) (
Figure 1b), whereas the 300 bp band was due to the similar primer and the 300 bp band sequences. Thus, the target sequence was mistaken for the primer sequence, and the overlapping sequence was eliminated from the synthesized sequence, resulting in three bands. Although two alternatively spliced variants of the yak’s
TLR4 gene were successfully cloned and identified in this study, it is still unclear if the yak’s
TLR4 gene contains other alternative splicing bodies, which demands an in-depth investigation.
As per the previous studies, the site of alternative splicing might impact the functionality of the encoded protein. Besides, altered peptide sequence also alters the ligand-binding sites, enzyme activity, allosteric regulation, or protein localization [
41]. 3D-model of the full-length TLR4 protein and a truncated TLR4 protein encoded by the exon-2 deleted
TLR4 transcript was constructed using the known protein structure and I-TASSER for the functional characterization of the TLR4 protein. The confidence of each model was quantitatively evaluated using the C-Score from the thread template alignment and the structural assembly simulation’s confluence parameters. Mostly, −5~2 C-Score indicates the high quality of the prediction model [
42]. TM-score, which measures the structural similarity between two structures, is mostly in the range of 0~1. TM-score > 0.5 indicates similar structures in the same SCOP/CATH folding family [
43]. Models with a C-Score > −1.5 and a TM-score > 0.5 have the correct folding structure. Based on the order of amino acids in the primary structure of the protein, TLR4-X1 contained three exons, encoding 841 amino acids. However, TLR4-X2 showed frameshift mutation in the open reading frame due to the deletion of exon-2. The translation was initiated at 434 bp of the CDS region, resulting in one low complexity region encoded by the first exon, four LRR domains were encoded by the second exon, and part of exon-3 was lost. 3D model of the TLR4 full length and truncated protein from exon-2 deleted
TLR4 transcript was predicted. The full-length protein showed C-Score: −0.09, TM-score: 0.72 ± 0.12 and the truncated protein showed C-Score: −0.23, TM-score: 0.68 ± 0.12. However, the C-Scores of both proteins were higher than −1.5. It suggested the high quality of both the TLR4 full-length protein and the truncated protein model. In addition, the template quality of the full-length TLR4 protein was slightly better than the truncated TLR4 protein. TM-scores of the two proteins were compared, which revealed that the TM-scores of both proteins were almost similar and > 0.5. It demonstrated the correct folding structure and high structural similarity between the protein models. The highest TM-score (3fxiA) of the TLR4 full-length protein matched the homologous template structure from the PDB library based on the amino acid sequence, and it corresponded to the human TLR4-human MD-2-
E. coli LPS Ra complex. The highest TM-score (3j0aA) of the truncated TLR4 protein matched with the TM-score of human Toll-like receptor 5. It suggested that the truncated protein contained frameshift mutation due to the deletion of exon-2 in the
TLR4 transcript, which resulted in the loss of some domains in the resulting protein and altered protein structure. The structure of the protein was closely correlated to its function. Deletion of exon-2 affected the protein function to some extent.
Functional bioinformatics outcome using ligand BioLiP template, which derived target protein binding site and enzyme classification, showed that the full-length TLR4 protein and IgA-specific serine endopeptidase were functionally identical. IgA-specific serine endopeptidase belongs to the IgA-specific serine endopeptidase family (S 6). Family S 6 contains identical enzymes from two gram-negative pathogenic bacteria,
Neisseria gonorrhoeae and
Haemophilus influenzae. These peptidases cleave the heavy chains of immunoglobulin A at prolyl bonds in the hinge region [
44], resulting in hydrolysis products consisting of the intact antigen-binding Fab and the Fc region of these antibody proteins [
45].
TLR4 truncated protein, S-acetyltransferase, and Oleoyl-hydrolase are functionally identical. In plastids, acyl-(acyl-carrier-protein) hydrolase (EC 3.1.2.14) releases fatty acids from the end-product of fatty acid synthesis, which forms glycerolipids in the cytoplasm. Two major processes that contribute to the biosynthesis of glycerolipids in plant cells are a) synthesis of fatty acids in plastids and b) incorporation of these fatty acids into glycerolipids in plastids and ER (endoplasmic reticula). Acyl-(acyl-carrier-protein) hydrolase (AH) plays a crucial role in the transportation of fatty acids from plastids to the cytoplasm for glycerolipid synthesis [
46]. In this study, the full-length TLR4 protein and truncated TLR4 protein showed altered structure and functions.
The phylogenetic tree constructed from the amino acid sequences of yak and other species established the close evolutionary relationship of yak with other cattle and distant evolutionary relationship with gorillas, in line with the previous studies. Amino acid sequence alignment of yak with other species elucidated 70–99% similarity between amino acid sequences from different species. It indicated that the
TLR4 gene, which plays crucial roles in animals, remained highly conserved throughout the evolutionary process. Previous studies have shown a close correlation between
TLR4 and diseases. For instance, missense mutations in
TLR4 were associated with decreased reactivity to LPS in a minority population [
47]. In chicken,
TLR4 polymorphism was associated with susceptibility to
Salmonella [
48]. It further demonstrated the correlation of the
TLR4 gene with immunity. Thus, in-depth research on the
TLR4 gene might improve the host’s resistance to multiple disorders.
TLR4 is primarily expressed by immune cells that participate in host defense, such as monocytes, macrophages, granulocytes, dendritic cells, lymphocytes, epithelial cells, endothelial cells, bone marrow monocytes, and so on [
49,
50,
51]. In this study, qRT-PCR was used to assess the expression levels of two alternatively spliced variants of the
TLR4 gene in seven distinct tissues. The results showed that
TLR4 gene expression levels were significantly different in different tissues, and it was highest in the spleen, followed by the lung. It is in line with previous studies in duck [
10], which showed specific
TLR4 gene expression and function. TLR4, a member of the PRR family, is a type I transmembrane glycoprotein. It participates in multiple functions, such as immune and inflammatory response [
52]. Independent samples t-test of the TLR4-X1 and TLR4-X2 demonstrated significantly different expression levels of TLR4-X1 and TLR4-X2 in the same tissue. It might be due to different immune responses induced by TLR4-X1 and TLR4-X2 in the yak.
TLR4 expression levels varied between the tissues to meet the functional requirements. In this study,
TLR4 gene expression in yak was found to be strictly regulated. It indicated that the alternative splicing of
TLR4 plays a crucial role in growth and development. The specific function of the two alternatively spliced
TLR4 will be explored in our future studies.