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Functional SNPs of INCENP Affect Semen Quality by Alternative Splicing Mode and Binding Affinity with the Target Bta-miR-378 in Chinese Holstein Bulls

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ABSTRACT

Inner centromere protein (INCENP) plays an important role in mitosis and meiosis as the main member of chromosomal passenger protein complex (CPC). To investigate the functional markers of the INCENP gene associated with semen quality, the single nucleotide polymorphisms (SNPs) g.19970 A>G and g.34078 T>G were identified and analyzed. The new splice variant INCENP-TV is characterized by the deletion of exon 12. The g.19970 A>G in the exonic splicing enhancer (ESE) motif region results in an aberrant splice variant by constructing two minigene expression vectors using the pSPL3 exon capturing vector and transfecting vectors into MLTC-1 cells. INCENP-TV was more highly expressed than INCENP-reference in adult bull testes. The g.34078 T>G located in the binding region of bta-miR-378 could affect the expression of INCENP, which was verified by luciferase assay. To analyze comprehensively the correlation of SNPs with sperm quality, haplotype combinations constructed by g.19970 A>G and g.34078 T>G, as well as g.-692 C>T and g.-556 G>T reported in our previous studies, were analyzed. The bulls with H1H12 and H2H2 exhibited a higher ejaculate volume than those with H2H10 and H9H12, respectively (P < 0.05). Bulls with H11H11 and H2H10 exhibited higher initial sperm motility than those with H2H2 (P < 0.05). The expression levels of INCENP in bulls with H1H12 and H11H11 were significantly higher than those in bulls with H9H12 (P < 0.05), as determined by qRT-PCR. Findings suggest that g.19970 A>G and g.34078 T>G in INCENP both of which appear to change the molecular and biological characteristics of the mRNA transcribed from the locus may serve as a biomarkers of male bovine fertility by affecting alternative splicing mode and binding affinity with the target bta-miR-378.

No MeSH data available.


Expression of haplotype combination of INCENP gross transcript (INCENP -G).The INCENP with the haplotype combinations H11H11 and H1H12 showed a significantly (P < 0.05) higher mRNA expression compared that with the haplotype combinations H2H10, H2H2 and H9H12. H1 = CGAT; H2 = CGAG; H9 = TTAT; H10 = TTAG; H11 = TTGT; H12 = TTGG. Means with different lowercase superscripts above the error bars are significantly different at P < 0.05.
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pone.0162730.g008: Expression of haplotype combination of INCENP gross transcript (INCENP -G).The INCENP with the haplotype combinations H11H11 and H1H12 showed a significantly (P < 0.05) higher mRNA expression compared that with the haplotype combinations H2H10, H2H2 and H9H12. H1 = CGAT; H2 = CGAG; H9 = TTAT; H10 = TTAG; H11 = TTGT; H12 = TTGG. Means with different lowercase superscripts above the error bars are significantly different at P < 0.05.

Mentions: Relative quantification was performed to illustrate the relative mRNA expression levels of INCENP gross transcript (INCENP-G) in the sperm cells from different haplotype combinations of bulls. The INCENP with the mutant haplotype H11H11 and H1H12 showed a significantly (P<0.05) higher mRNA expression compared with the haplotype combinations H2H10, H2H2, and H9H12 (Fig 8). Therefore, the SNPs, g.19970 A>G, g.34078 T>G, g.-692 C>T, and g.-556 G>T can potentially influence the mRNA expression of INCENP.


Functional SNPs of INCENP Affect Semen Quality by Alternative Splicing Mode and Binding Affinity with the Target Bta-miR-378 in Chinese Holstein Bulls
Expression of haplotype combination of INCENP gross transcript (INCENP -G).The INCENP with the haplotype combinations H11H11 and H1H12 showed a significantly (P < 0.05) higher mRNA expression compared that with the haplotype combinations H2H10, H2H2 and H9H12. H1 = CGAT; H2 = CGAG; H9 = TTAT; H10 = TTAG; H11 = TTGT; H12 = TTGG. Means with different lowercase superscripts above the error bars are significantly different at P < 0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036895&req=5

pone.0162730.g008: Expression of haplotype combination of INCENP gross transcript (INCENP -G).The INCENP with the haplotype combinations H11H11 and H1H12 showed a significantly (P < 0.05) higher mRNA expression compared that with the haplotype combinations H2H10, H2H2 and H9H12. H1 = CGAT; H2 = CGAG; H9 = TTAT; H10 = TTAG; H11 = TTGT; H12 = TTGG. Means with different lowercase superscripts above the error bars are significantly different at P < 0.05.
Mentions: Relative quantification was performed to illustrate the relative mRNA expression levels of INCENP gross transcript (INCENP-G) in the sperm cells from different haplotype combinations of bulls. The INCENP with the mutant haplotype H11H11 and H1H12 showed a significantly (P<0.05) higher mRNA expression compared with the haplotype combinations H2H10, H2H2, and H9H12 (Fig 8). Therefore, the SNPs, g.19970 A>G, g.34078 T>G, g.-692 C>T, and g.-556 G>T can potentially influence the mRNA expression of INCENP.

View Article: PubMed Central - PubMed

ABSTRACT

Inner centromere protein (INCENP) plays an important role in mitosis and meiosis as the main member of chromosomal passenger protein complex (CPC). To investigate the functional markers of the INCENP gene associated with semen quality, the single nucleotide polymorphisms (SNPs) g.19970 A&gt;G and g.34078 T&gt;G were identified and analyzed. The new splice variant INCENP-TV is characterized by the deletion of exon 12. The g.19970 A&gt;G in the exonic splicing enhancer (ESE) motif region results in an aberrant splice variant by constructing two minigene expression vectors using the pSPL3 exon capturing vector and transfecting vectors into MLTC-1 cells. INCENP-TV was more highly expressed than INCENP-reference in adult bull testes. The g.34078 T&gt;G located in the binding region of bta-miR-378 could affect the expression of INCENP, which was verified by luciferase assay. To analyze comprehensively the correlation of SNPs with sperm quality, haplotype combinations constructed by g.19970 A&gt;G and g.34078 T&gt;G, as well as g.-692 C&gt;T and g.-556 G&gt;T reported in our previous studies, were analyzed. The bulls with H1H12 and H2H2 exhibited a higher ejaculate volume than those with H2H10 and H9H12, respectively (P &lt; 0.05). Bulls with H11H11 and H2H10 exhibited higher initial sperm motility than those with H2H2 (P &lt; 0.05). The expression levels of INCENP in bulls with H1H12 and H11H11 were significantly higher than those in bulls with H9H12 (P &lt; 0.05), as determined by qRT-PCR. Findings suggest that g.19970 A&gt;G and g.34078 T&gt;G in INCENP both of which appear to change the molecular and biological characteristics of the mRNA transcribed from the locus may serve as a biomarkers of male bovine fertility by affecting alternative splicing mode and binding affinity with the target bta-miR-378.

No MeSH data available.