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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

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>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.


The single-nucleotide polymorphism (g.34078 T>G) located in the seed region of bta-miR-378 binding to the 3′-UTR of the bovine INCENP gene.(A) The bta-miR-378: mRNA of INCENP interaction is shown. The altered allele is highlighted. (B) Computational modeling of the interaction between miR-378 and 3’-UTR that contains g.34078 T>G-T genotype (wild type) or g.34078 T>G-G (mutant type) was performed on RNAHYBRID software online.
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pone.0162730.g006: The single-nucleotide polymorphism (g.34078 T>G) located in the seed region of bta-miR-378 binding to the 3′-UTR of the bovine INCENP gene.(A) The bta-miR-378: mRNA of INCENP interaction is shown. The altered allele is highlighted. (B) Computational modeling of the interaction between miR-378 and 3’-UTR that contains g.34078 T>G-T genotype (wild type) or g.34078 T>G-G (mutant type) was performed on RNAHYBRID software online.

Mentions: Generally, specificity of the target recognition of miRNAs is crucially dependent on the seed region of the miRNAs regulatory region (nucleotides 2–7 of the 5'-end). Variants, such as SNPs in the miRNA binding site, especially in the seed region, may result in the variation of mRNA or protein levels in organizations. Using RNA22, a method for identifying microRNA binding sites and their corresponding heteroduplexes, we predicted that bta-miR-378 could bind to the 3'UTR of INCENP when g.34078 T mutated into G. Furthermore, we predicted the minimum free energy hybridization by RNAhybrid, and the result showed that the minimum free energy of mutant type was lower than wild type, presenting that binding capacity of mutant type and bta-miR-378 was stronger than wild type. All results suggested that g.34078 T>G could influence the association of bta-miR-378 and 3'UTR of INCENP (Fig 6). Huang et al. found that bta-miR-378 was highly expressed in the testis by Q-PCR and combined Solexa sequencing with bioinformatics [27].


Functional SNPs of INCENP Affect Semen Quality by Alternative Splicing Mode and Binding Affinity with the Target Bta-miR-378 in Chinese Holstein Bulls
The single-nucleotide polymorphism (g.34078 T>G) located in the seed region of bta-miR-378 binding to the 3′-UTR of the bovine INCENP gene.(A) The bta-miR-378: mRNA of INCENP interaction is shown. The altered allele is highlighted. (B) Computational modeling of the interaction between miR-378 and 3’-UTR that contains g.34078 T>G-T genotype (wild type) or g.34078 T>G-G (mutant type) was performed on RNAHYBRID software online.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0162730.g006: The single-nucleotide polymorphism (g.34078 T>G) located in the seed region of bta-miR-378 binding to the 3′-UTR of the bovine INCENP gene.(A) The bta-miR-378: mRNA of INCENP interaction is shown. The altered allele is highlighted. (B) Computational modeling of the interaction between miR-378 and 3’-UTR that contains g.34078 T>G-T genotype (wild type) or g.34078 T>G-G (mutant type) was performed on RNAHYBRID software online.
Mentions: Generally, specificity of the target recognition of miRNAs is crucially dependent on the seed region of the miRNAs regulatory region (nucleotides 2–7 of the 5'-end). Variants, such as SNPs in the miRNA binding site, especially in the seed region, may result in the variation of mRNA or protein levels in organizations. Using RNA22, a method for identifying microRNA binding sites and their corresponding heteroduplexes, we predicted that bta-miR-378 could bind to the 3'UTR of INCENP when g.34078 T mutated into G. Furthermore, we predicted the minimum free energy hybridization by RNAhybrid, and the result showed that the minimum free energy of mutant type was lower than wild type, presenting that binding capacity of mutant type and bta-miR-378 was stronger than wild type. All results suggested that g.34078 T>G could influence the association of bta-miR-378 and 3'UTR of INCENP (Fig 6). Huang et al. found that bta-miR-378 was highly expressed in the testis by Q-PCR and combined Solexa sequencing with bioinformatics [27].

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>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.