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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.


Identification of INCENP -TV and SNP g.19970 A>G and PCR-RFLP of of bovine INCENP gene.(A) The cloning sequencing result of INCENP-cDNA by a pair of primers, F4 and R4. A new transcript INCENP -TV which deleted 12 bp was found. (B) Genomic structure of the bovine INCENP gene which consists of 20 exons, comprising 2646 bp of coding sequence. (C) The splicing pattern of the INCENP-TV splice variant which deletes exon 12, merely composed of 12 bp. (D) The SNP g.19970 A>G (rs:109416157) which lies in the region of intron 11 was discovered by sequencing (E) PCR-RFLP was employed to detect the mutation by Nde I. The products of endonuclease digestion were tested in 2.5% agarose gel, showing three genotypes: AA (606 +110 bp), AG (716 + 606 + 110 bp) and GG (716 bp).
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pone.0162730.g001: Identification of INCENP -TV and SNP g.19970 A>G and PCR-RFLP of of bovine INCENP gene.(A) The cloning sequencing result of INCENP-cDNA by a pair of primers, F4 and R4. A new transcript INCENP -TV which deleted 12 bp was found. (B) Genomic structure of the bovine INCENP gene which consists of 20 exons, comprising 2646 bp of coding sequence. (C) The splicing pattern of the INCENP-TV splice variant which deletes exon 12, merely composed of 12 bp. (D) The SNP g.19970 A>G (rs:109416157) which lies in the region of intron 11 was discovered by sequencing (E) PCR-RFLP was employed to detect the mutation by Nde I. The products of endonuclease digestion were tested in 2.5% agarose gel, showing three genotypes: AA (606 +110 bp), AG (716 + 606 + 110 bp) and GG (716 bp).

Mentions: Based on specific primers (S1 Table) for INCENP cDNA, PCR amplification using various tissue cDNA as a template was performed. These products were purified, cloned, and sequenced (Fig 1A). One novel INCENP gene transcript variant (INCENP-TV) was found in various tissues referring to transcript of INCENP reference (GenBank accession number: XM_002707799.3). The sequence alignment results indicated that the splice variant INCENP-TV lacked the 12th exon, which contained 12 bp (Fig 1C). The sequence of INCENP-TV was submitted to the National Center of Biotechnology Information (GenBank accession number: KU499879).


Functional SNPs of INCENP Affect Semen Quality by Alternative Splicing Mode and Binding Affinity with the Target Bta-miR-378 in Chinese Holstein Bulls
Identification of INCENP -TV and SNP g.19970 A>G and PCR-RFLP of of bovine INCENP gene.(A) The cloning sequencing result of INCENP-cDNA by a pair of primers, F4 and R4. A new transcript INCENP -TV which deleted 12 bp was found. (B) Genomic structure of the bovine INCENP gene which consists of 20 exons, comprising 2646 bp of coding sequence. (C) The splicing pattern of the INCENP-TV splice variant which deletes exon 12, merely composed of 12 bp. (D) The SNP g.19970 A>G (rs:109416157) which lies in the region of intron 11 was discovered by sequencing (E) PCR-RFLP was employed to detect the mutation by Nde I. The products of endonuclease digestion were tested in 2.5% agarose gel, showing three genotypes: AA (606 +110 bp), AG (716 + 606 + 110 bp) and GG (716 bp).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0162730.g001: Identification of INCENP -TV and SNP g.19970 A>G and PCR-RFLP of of bovine INCENP gene.(A) The cloning sequencing result of INCENP-cDNA by a pair of primers, F4 and R4. A new transcript INCENP -TV which deleted 12 bp was found. (B) Genomic structure of the bovine INCENP gene which consists of 20 exons, comprising 2646 bp of coding sequence. (C) The splicing pattern of the INCENP-TV splice variant which deletes exon 12, merely composed of 12 bp. (D) The SNP g.19970 A>G (rs:109416157) which lies in the region of intron 11 was discovered by sequencing (E) PCR-RFLP was employed to detect the mutation by Nde I. The products of endonuclease digestion were tested in 2.5% agarose gel, showing three genotypes: AA (606 +110 bp), AG (716 + 606 + 110 bp) and GG (716 bp).
Mentions: Based on specific primers (S1 Table) for INCENP cDNA, PCR amplification using various tissue cDNA as a template was performed. These products were purified, cloned, and sequenced (Fig 1A). One novel INCENP gene transcript variant (INCENP-TV) was found in various tissues referring to transcript of INCENP reference (GenBank accession number: XM_002707799.3). The sequence alignment results indicated that the splice variant INCENP-TV lacked the 12th exon, which contained 12 bp (Fig 1C). The sequence of INCENP-TV was submitted to the National Center of Biotechnology Information (GenBank accession number: KU499879).

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.