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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.


Exonic splice enhancer (ESE) motif threshold scores associated with INCENP genotypes.Bar graphs represent scores above the threshold for the ESE motifs within the A or G allele in locus g.19970 A>G. The red square indicates that the introduction of allele G, relative to allele A in locus g.19970 A>G, increased three binding sites of the auxiliary splicing proteins SRSF1, SRSF1 (IgM-BRCA1) and SRSF5 and deleted one of SRSF6.
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pone.0162730.g003: Exonic splice enhancer (ESE) motif threshold scores associated with INCENP genotypes.Bar graphs represent scores above the threshold for the ESE motifs within the A or G allele in locus g.19970 A>G. The red square indicates that the introduction of allele G, relative to allele A in locus g.19970 A>G, increased three binding sites of the auxiliary splicing proteins SRSF1, SRSF1 (IgM-BRCA1) and SRSF5 and deleted one of SRSF6.

Mentions: In view of the discovery and functional understanding of SNP, ESEfinder predicted that the g.19970 A>G located in an exonic splice enhancer (ESE) motif region and mutation added several binding sites for the splicing factors SRSF1, SRSF1 (IgM-BRCA1), SRSF5, and SRSF6 (Fig 3). Thus, we suggest that SNP (g.19970 A>G) may be correlated with the presence or absence of the splice variant INCENP-TV. The mutation appears to strengthen the role of the constitutive acceptor splice site, which led to INCENP-TV splicing change in mature mRNA during INCENP transcription.


Functional SNPs of INCENP Affect Semen Quality by Alternative Splicing Mode and Binding Affinity with the Target Bta-miR-378 in Chinese Holstein Bulls
Exonic splice enhancer (ESE) motif threshold scores associated with INCENP genotypes.Bar graphs represent scores above the threshold for the ESE motifs within the A or G allele in locus g.19970 A>G. The red square indicates that the introduction of allele G, relative to allele A in locus g.19970 A>G, increased three binding sites of the auxiliary splicing proteins SRSF1, SRSF1 (IgM-BRCA1) and SRSF5 and deleted one of SRSF6.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0162730.g003: Exonic splice enhancer (ESE) motif threshold scores associated with INCENP genotypes.Bar graphs represent scores above the threshold for the ESE motifs within the A or G allele in locus g.19970 A>G. The red square indicates that the introduction of allele G, relative to allele A in locus g.19970 A>G, increased three binding sites of the auxiliary splicing proteins SRSF1, SRSF1 (IgM-BRCA1) and SRSF5 and deleted one of SRSF6.
Mentions: In view of the discovery and functional understanding of SNP, ESEfinder predicted that the g.19970 A>G located in an exonic splice enhancer (ESE) motif region and mutation added several binding sites for the splicing factors SRSF1, SRSF1 (IgM-BRCA1), SRSF5, and SRSF6 (Fig 3). Thus, we suggest that SNP (g.19970 A>G) may be correlated with the presence or absence of the splice variant INCENP-TV. The mutation appears to strengthen the role of the constitutive acceptor splice site, which led to INCENP-TV splicing change in mature mRNA during INCENP transcription.

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.