Identification and quantitative mRNA analysis of a novel splice variant of GPIHBP1 in dairy cattle

Abstract

Background: Identification of functional genes affecting milk production traits is very crucial for improving breeding efficiency in dairy cattle. Many potential candidate genes have been identified through our previous genome wide association study (GWAS). Of these, GPIHBP1 is an important novel candidate gene for milk production traits. However, the mRNA structure of the bovine GPIHBP1 gene is not fully determined up to now.

Results: In this study, we identified a novel alternatively splice transcript variant (X5) which leads to a 31 bp insertion in exon 3 and also confirmed the other four existed transcripts (X1, X2, X3 and X4) of the bovine GPIHBP1 gene. We showed that transcript X5 with a 31 bp insertion and transcript X1 with an 8 bp deletion might have tremendous effect on the protein function and structure of GPIHBP1, respectively. With semi-quantitative PCR and quantitative real-time RT-PCR, we found that the mRNA expression of GPIHBP1, GPIHBP1-X1 and GPIHBP1-X5 in mammary gland of lactating cows were much higher than that in other tissues.

Conclusions: Our study reports a novel alternative splicing of GPIHBP1 in bovine for the first time and provide useful information for the further functional analyses of GPIHBP1 in dairy cattle.

Keywords: Alternative splice variant; Cattle; Expression pattern; GPIHBP1.

Figure 1

PCR analysis of the GPIHBP1 coding region in samples of mammary gland using three pairs of primers. (A) With primer 1, transcript X2 (603 bp), transcript X3 (289 bp) and transcript X4 (192 bp) were observed as expected (according to the mRNA sequence of the bovine GPIHBP1 gene in NCBI database). M1: DL2000, 2,000 bp, 1,000 bp, 750 bp, 500 bp, 250 bp, 100 bp. (B) With primer 2, in addition to the expected PCR band (352 bp and 344 bp), a band of 383 bp was also observed in all samples. M2: 100 bp DNA Ladder from 1,500–100 bp. (C) With primer 3, only the expected band (388 bp) was observed in all samples. M3: DL500, 500 bp, 400 bp, 300 bp, 200 bp, 150 bp, 100 bp and 50 bp.

Figure 2

Schematic representation of the GPIHBP1 alternative splice variants. Transcripts X1 (XM_002692563.2), X2 (XM_005215283.1), X3 (XM_005215284.1) and X4 (XM_005215285.1) were obtained from NCBI. Transcript X5 was observed by RNA-seq (unpublished data). Transcript X1 contained a 8 bp deletion in exon 3. Transcript X5 contained a 31 bp insertion in exon 3. Although transcript X2, X3 and X4 had different 5′ untranslated region, they had the same translation initiation site (TIS).

Figure 3

Predicted secondary structures of bovine and human GPIHBP1. Residues involved in the N-signal peptide formation are indicated by underline, α-helices are in red, β-sheets are in green, GPI-modification site is in blue, alternative GPI-modification site is in purple. (A) Amino acid sequence alignments of bovine and human GPIHBP1. The predicted secondary structures for bovine-p1 (XP_002692609.2) and bovine-p2 (XP_005215340.1, XP_005215341.1 and XP_005215342.1) are similar for several regions with that of human GPIHBP1. (B) The predicted secondary structures for bovine-p5.1, bovine-p5.2, bovine-p5.3, bovine-p5.4 and bovine-p5.5 of transcript X5.

Figure 4

Predicted tertiary structures of bovine and human GPIHBP1. The reported human CD59 was used as the reference to obtain predicted GPIHBP1 tertiary structures by the SWISS MODEL method. The rainbow color code describes the tertiary structures from the N-termini (blue) to C-termini (red) for GPIHBP1 UPAR/Ly6 domains. Arrows indicate the directions for β-sheets.

Figure 5

The mRNA expression patterns of GPIHBP1 , GPIHBP1 -X1 and GPIHBP1 -X5 revealed by RT-PCR. The histograms represent the mRNA expression level of GPIHBP1, GPIHBP1-X1 and GPIHBP1-X5 in eight tissues of three cows. mRNA expression levels in mammary gland of GPIHBP1, GPIHBP1-X1 and GPIHBP1-X5 all were highest among 8 tissues. The different capital letters indicated significant differences in the expression among eight tissues at P <0.01.