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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 exon trapping vector pSPL3 used to assay SNP g.19970 A>G function.(A) The pSPL3 vector contains splice donor (SD) and splice acceptor (SA) sites that operate as exons, and a functional intron, with transcription beginning following the SV40 promoter and ending at the LPAS (late poly (A) signal). Wild-type segment and mutant segment containing 292 bp of intron 11, 12 bp of exon 12, 412 bp of intron 12 and harboring either the A or G alleles were separately cloned into the EcoRI and XhoI cloning sites of the pSPL3 vector. Two minigene expression vectors were transiently transfected into MLTC-1 cells. (B) RT-PCR analysis of the INCENP spliced transcripts on 3% agarose gel. Due to the difference of only 12 bp, the result of electrophoresis is not obvious. (C) cDNA was sequenced and the result shown that mutant and empty pSPL3-control expressed a fragment of 263 bp. (D) Cloning sequencing was implemented to verify the fragment of WT. The result showed that WT obtained 275 bp and 263 bp PCR fragment. The sizes of the fragment (275 bp) corresponded to the amplification exon 12 (12 bp) and pSPL3 control plasmid (263 bp). The size of the fragment (263 bp) corresponded to the pSPL3 control plasmid (263 bp).
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pone.0162730.g004: The exon trapping vector pSPL3 used to assay SNP g.19970 A>G function.(A) The pSPL3 vector contains splice donor (SD) and splice acceptor (SA) sites that operate as exons, and a functional intron, with transcription beginning following the SV40 promoter and ending at the LPAS (late poly (A) signal). Wild-type segment and mutant segment containing 292 bp of intron 11, 12 bp of exon 12, 412 bp of intron 12 and harboring either the A or G alleles were separately cloned into the EcoRI and XhoI cloning sites of the pSPL3 vector. Two minigene expression vectors were transiently transfected into MLTC-1 cells. (B) RT-PCR analysis of the INCENP spliced transcripts on 3% agarose gel. Due to the difference of only 12 bp, the result of electrophoresis is not obvious. (C) cDNA was sequenced and the result shown that mutant and empty pSPL3-control expressed a fragment of 263 bp. (D) Cloning sequencing was implemented to verify the fragment of WT. The result showed that WT obtained 275 bp and 263 bp PCR fragment. The sizes of the fragment (275 bp) corresponded to the amplification exon 12 (12 bp) and pSPL3 control plasmid (263 bp). The size of the fragment (263 bp) corresponded to the pSPL3 control plasmid (263 bp).

Mentions: To determine whether the internal mutation g.19970 A>G in intron 11 leads to an increase in the predicted SR protein motifs and causes diversity of mRNA, the principle of alternative splicing was assessed using minigene splicing assay. We amplified 716 bp genomic fragments, including wild-type g.19970 A>G-A (abbreviated as WT) and mutant g.19970 A>G-G (abbreviated as mutant) into the pSPL3 vector (Fig 4A).


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 exon trapping vector pSPL3 used to assay SNP g.19970 A>G function.(A) The pSPL3 vector contains splice donor (SD) and splice acceptor (SA) sites that operate as exons, and a functional intron, with transcription beginning following the SV40 promoter and ending at the LPAS (late poly (A) signal). Wild-type segment and mutant segment containing 292 bp of intron 11, 12 bp of exon 12, 412 bp of intron 12 and harboring either the A or G alleles were separately cloned into the EcoRI and XhoI cloning sites of the pSPL3 vector. Two minigene expression vectors were transiently transfected into MLTC-1 cells. (B) RT-PCR analysis of the INCENP spliced transcripts on 3% agarose gel. Due to the difference of only 12 bp, the result of electrophoresis is not obvious. (C) cDNA was sequenced and the result shown that mutant and empty pSPL3-control expressed a fragment of 263 bp. (D) Cloning sequencing was implemented to verify the fragment of WT. The result showed that WT obtained 275 bp and 263 bp PCR fragment. The sizes of the fragment (275 bp) corresponded to the amplification exon 12 (12 bp) and pSPL3 control plasmid (263 bp). The size of the fragment (263 bp) corresponded to the pSPL3 control plasmid (263 bp).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0162730.g004: The exon trapping vector pSPL3 used to assay SNP g.19970 A>G function.(A) The pSPL3 vector contains splice donor (SD) and splice acceptor (SA) sites that operate as exons, and a functional intron, with transcription beginning following the SV40 promoter and ending at the LPAS (late poly (A) signal). Wild-type segment and mutant segment containing 292 bp of intron 11, 12 bp of exon 12, 412 bp of intron 12 and harboring either the A or G alleles were separately cloned into the EcoRI and XhoI cloning sites of the pSPL3 vector. Two minigene expression vectors were transiently transfected into MLTC-1 cells. (B) RT-PCR analysis of the INCENP spliced transcripts on 3% agarose gel. Due to the difference of only 12 bp, the result of electrophoresis is not obvious. (C) cDNA was sequenced and the result shown that mutant and empty pSPL3-control expressed a fragment of 263 bp. (D) Cloning sequencing was implemented to verify the fragment of WT. The result showed that WT obtained 275 bp and 263 bp PCR fragment. The sizes of the fragment (275 bp) corresponded to the amplification exon 12 (12 bp) and pSPL3 control plasmid (263 bp). The size of the fragment (263 bp) corresponded to the pSPL3 control plasmid (263 bp).
Mentions: To determine whether the internal mutation g.19970 A>G in intron 11 leads to an increase in the predicted SR protein motifs and causes diversity of mRNA, the principle of alternative splicing was assessed using minigene splicing assay. We amplified 716 bp genomic fragments, including wild-type g.19970 A>G-A (abbreviated as WT) and mutant g.19970 A>G-G (abbreviated as mutant) into the pSPL3 vector (Fig 4A).

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.