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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.


INCENP gene structure, location of g.34078 T>G, sequencing results and band patterns of the three genotypes g.34078 T>G.Part A showed Bovine INCENP gene structure, location, and results of sequencing of g.34078 T>G. B: 2.5% agarose gel showing band patterns of SNP g.34078 T>G digested with Alu I. Digestions of PCR products of INCENP g.34078 T>G locus with Alu I produced 205-bp bands for homozygous genotype TT, 205-, 126-, and 79-bp bands for heterozygous genotype TG, 126- and 79-bp bands for homozygous genotype GG.
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pone.0162730.g005: INCENP gene structure, location of g.34078 T>G, sequencing results and band patterns of the three genotypes g.34078 T>G.Part A showed Bovine INCENP gene structure, location, and results of sequencing of g.34078 T>G. B: 2.5% agarose gel showing band patterns of SNP g.34078 T>G digested with Alu I. Digestions of PCR products of INCENP g.34078 T>G locus with Alu I produced 205-bp bands for homozygous genotype TT, 205-, 126-, and 79-bp bands for heterozygous genotype TG, 126- and 79-bp bands for homozygous genotype GG.

Mentions: MiRNAs are post-transcriptional regulators that bind primarily to the 3′-untranslated region (UTR) of the target mRNAs. The SNPs in the binding site of miRNA, namely 3'UTR of the targeted mRNA, have been identified as regulatory mechanism of targeted mRNA. In order to verify SNPs in 3'UTR of INCENP gene, we detected the polymorphism of 3'UTR. Approximately 637 bp of 3'UTR and partial exon 20 was cloned and screened using a bi-directional DNA sequencing approach to identify whether SNP existed in the 3'UTR of the INCENP gene. Only one SNP (g.34078 T>G, rs: 42658780) was found using the INCENP gene sequence (GenBank accession number: AC_000186.1) as reference (Fig 5A). Genotyping was then performed by PCR-RFLP with Alu I. The products of endonuclease digestion were tested on 2.5% agarose gel, showing three genotypes: TT (205 bp), TG (205 + 126 + 79 bp), and GG (126 +79 bp) (Fig 5B).


Functional SNPs of INCENP Affect Semen Quality by Alternative Splicing Mode and Binding Affinity with the Target Bta-miR-378 in Chinese Holstein Bulls
INCENP gene structure, location of g.34078 T>G, sequencing results and band patterns of the three genotypes g.34078 T>G.Part A showed Bovine INCENP gene structure, location, and results of sequencing of g.34078 T>G. B: 2.5% agarose gel showing band patterns of SNP g.34078 T>G digested with Alu I. Digestions of PCR products of INCENP g.34078 T>G locus with Alu I produced 205-bp bands for homozygous genotype TT, 205-, 126-, and 79-bp bands for heterozygous genotype TG, 126- and 79-bp bands for homozygous genotype GG.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0162730.g005: INCENP gene structure, location of g.34078 T>G, sequencing results and band patterns of the three genotypes g.34078 T>G.Part A showed Bovine INCENP gene structure, location, and results of sequencing of g.34078 T>G. B: 2.5% agarose gel showing band patterns of SNP g.34078 T>G digested with Alu I. Digestions of PCR products of INCENP g.34078 T>G locus with Alu I produced 205-bp bands for homozygous genotype TT, 205-, 126-, and 79-bp bands for heterozygous genotype TG, 126- and 79-bp bands for homozygous genotype GG.
Mentions: MiRNAs are post-transcriptional regulators that bind primarily to the 3′-untranslated region (UTR) of the target mRNAs. The SNPs in the binding site of miRNA, namely 3'UTR of the targeted mRNA, have been identified as regulatory mechanism of targeted mRNA. In order to verify SNPs in 3'UTR of INCENP gene, we detected the polymorphism of 3'UTR. Approximately 637 bp of 3'UTR and partial exon 20 was cloned and screened using a bi-directional DNA sequencing approach to identify whether SNP existed in the 3'UTR of the INCENP gene. Only one SNP (g.34078 T>G, rs: 42658780) was found using the INCENP gene sequence (GenBank accession number: AC_000186.1) as reference (Fig 5A). Genotyping was then performed by PCR-RFLP with Alu I. The products of endonuclease digestion were tested on 2.5% agarose gel, showing three genotypes: TT (205 bp), TG (205 + 126 + 79 bp), and GG (126 +79 bp) (Fig 5B).

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.