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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.


Luciferase activity of different genotypes at g.34078 T>G locus showing PMIR-3′-UTR-T (wild type) or PMIR-3′-UTR-G (mutant type) construct co-transfected with miR-378.Data are from the three transfection experiments with assays performed in six replications. CmiR001-MR04 serves as a scrambled control. Firefly luciferase activity was normalized to the absorbance value of pRL-TK. Vertical bars represent the SE of six replication experiments. Means with different lowercase superscripts above the error bars are significantly different at P < 0.05.
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pone.0162730.g007: Luciferase activity of different genotypes at g.34078 T>G locus showing PMIR-3′-UTR-T (wild type) or PMIR-3′-UTR-G (mutant type) construct co-transfected with miR-378.Data are from the three transfection experiments with assays performed in six replications. CmiR001-MR04 serves as a scrambled control. Firefly luciferase activity was normalized to the absorbance value of pRL-TK. Vertical bars represent the SE of six replication experiments. Means with different lowercase superscripts above the error bars are significantly different at P < 0.05.

Mentions: To verify whether bta-miR-378 binds with the 3'-UTR of INCENP or the SNP g.34078 T>G influences differential binding affinity, the luciferase reporter assay was implemented. The results revealed that the bta-miR-378 reduced the luciferase reporter gene activity, indicating that bta-miR-378 suppressed expression of reporter gene. This seems to be different from forecasting results of RNA22. Nevertheless, the data also showed that expression of reporter gene of mutant type 3'-UTR was below wild type (P < 0.05), suggesting a higher binding affinity for the mutant type 3'-UTR compared with the wild-type 3'-UTR, which is consistent with the computational prediction results (Fig 7).


Functional SNPs of INCENP Affect Semen Quality by Alternative Splicing Mode and Binding Affinity with the Target Bta-miR-378 in Chinese Holstein Bulls
Luciferase activity of different genotypes at g.34078 T>G locus showing PMIR-3′-UTR-T (wild type) or PMIR-3′-UTR-G (mutant type) construct co-transfected with miR-378.Data are from the three transfection experiments with assays performed in six replications. CmiR001-MR04 serves as a scrambled control. Firefly luciferase activity was normalized to the absorbance value of pRL-TK. Vertical bars represent the SE of six replication experiments. 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.g007: Luciferase activity of different genotypes at g.34078 T>G locus showing PMIR-3′-UTR-T (wild type) or PMIR-3′-UTR-G (mutant type) construct co-transfected with miR-378.Data are from the three transfection experiments with assays performed in six replications. CmiR001-MR04 serves as a scrambled control. Firefly luciferase activity was normalized to the absorbance value of pRL-TK. Vertical bars represent the SE of six replication experiments. Means with different lowercase superscripts above the error bars are significantly different at P < 0.05.
Mentions: To verify whether bta-miR-378 binds with the 3'-UTR of INCENP or the SNP g.34078 T>G influences differential binding affinity, the luciferase reporter assay was implemented. The results revealed that the bta-miR-378 reduced the luciferase reporter gene activity, indicating that bta-miR-378 suppressed expression of reporter gene. This seems to be different from forecasting results of RNA22. Nevertheless, the data also showed that expression of reporter gene of mutant type 3'-UTR was below wild type (P < 0.05), suggesting a higher binding affinity for the mutant type 3'-UTR compared with the wild-type 3'-UTR, which is consistent with the computational prediction results (Fig 7).

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.