Multiple promoters and targeted microRNAs direct the expressions of HMGB3 gene transcripts in dairy cattle.

HMGB3 (high-mobility group box 3) is an X-linked member of a family of sequence-independent chromatin-binding proteins and functions as a universal sentinel for nucleic acid-mediated innate immune responses. The splice variant expression, promoter characterization and targeted microRNAs of the bovine HMGB3 gene were investigated to explore its expression pattern and possible regulatory mechanism. The results revealed that the expression of HMGB3 transcript variants 1 and 2 (HMGB3-TV1 and HMGB3-TV2) mRNA in the mastitis-infected mammary gland tissues was up-regulated by 8.46- and 5.31-fold respectively compared with that in healthy tissues (P < 0.05). HMGB3-TV1 was highly expressed in the mammary gland tissues, whereas HMGB3-TV2 was expressed primarily in liver. Functional analyses indicated that HMGB3 transcription is regulated by three distinct promoters - promoters 1, 2 and 3 (P1, P2 and P3) - resulting in two alternative transcripts with the same 3'-untranslated region. Promoter luciferase activity analysis suggested that the core sequences of P1 and P2 were mapped in the region of g.1535 to ~g.2076 and g.2074 to ~g.2491 respectively. The g.5880C>T SNP in P3 affected its base promoter activity, and different genotypes were associated with the bovine somatic count score. The expression of targets bovine miR-17-5p, miR-20b and miR-93 of the HMGB3 gene was down-regulated 1.56-, 1.72- and 2.94-fold respectively in mammary gland tissues as compared with that in healthy tissues (P < 0.05). The findings suggest that HMGB3 expression is under complex transcriptional and post-transcriptional control by alternate promoter usage, alternative splicing mechanism and microRNAs in dairy cattle.

Alternative splicing and mRNA expression analysis of bovine SLAMF7 gene in healthy and mastitis mammary tissues.

The signaling lymphocyte-activating molecule family 7 (SLAMF7) proteins serve as adhesion molecules on the surface of a variety of mature hematopoietic cells, and also partially control certain innate and adaptive immune responses. We characterized three novel bovine SLAMF7 splice variants, designated as SLAMF7-AS1, AS2, and AS3. All three novel SLAMF7 isoforms are derived from the complete transcripts (SLAMF7-complete) via alternative splicing (AS). The patterns of the three splice variants are exon skipping and alternative 5' splice sites. Bovine SLAMF7 transcripts are expressed in mammary tissue, as demonstrated by real-time PCR. The levels of the complete transcript expression in the normal mammary tissues were higher than that in Staphylococcus aureus (Staph. aureus)-induced mastitis mammary tissues. However, it was not significant for the mRNA expression level comparison between these two kinds of mammary. The SLAMF-AS2 isoforms are expressed the lowest levels among the three transcripts in both normal and infected mammary tissues. This study provides clues for a better understanding of bovine SLAMF7 gene function.

Identification of splice variants, targeted microRNAs and functional single nucleotide polymorphisms of the BOLA-DQA2 gene in dairy cattle.

Major histocompatibility complex, class II, DQ alpha 2, also named BOLA-DQA2, belongs to the Bovine Leukocyte Antigen (BOLA) class II genes which are involved in the immune response. To explore the variability of the BOLA-DQA2 gene and resistance to mastitis in cows, the splice variants (SV), targeted microRNAs (miRNAs), and single nucleotide polymorphisms (SNPs) were identified in this study. A new SV (BOLA-DQA2-SV1) lacking part of exon 3 (195 bp) and two 3'-untranslated regions (UTR) (52 bp+167 bp) of the BOLA-DQA2 gene was found in the healthy and mastitis-infected mammary gland tissues. Four of 13 new SNPs and multiple nucleotide polymorphisms resulted in amino acid changes in the protein and SNP (c. +1283 C>T) may affect the binding to the seed sequence of bta-miR-2318. Further, we detected the relative expressions of two BOLA-DQA2 transcripts and five candidated microRNAs binding to the 3'-UTR of two transcripts in the mammary gland tissues in dairy cattle by using the quantitative real-time polymerase chain reaction. The result showed that expression of the BOLA-DQA2-SV1 mRNA was significantly upregulated 2.67-fold (p<0.05) in mastitis-infected mammary tissues (n = 5) compared with the healthy mammary gland mammary tissues (n = 5). Except for bta-miR-1777a, miRNA expression (bta-miR-296, miR-2430, and miR-671) was upregulated 1.75 to 2.59-fold (p<0.05), whereas miR-2318 was downregulated in the mastitis cows. Our findings reveal that BOLA-DQA2-SV1 may play an important role in the mastitis resistance in dairy cattle. Whether the SNPs affect the structure of the BOLA-DQA2 gene or association with mastitis resistance is unknown and warrants further investigation.

Solexa sequencing of novel and differentially expressed microRNAs in testicular and ovarian tissues in Holstein cattle.

The posttranscriptional gene regulation mediated by microRNA plays an important role in the development and function of male and female reproductive organs and germ cells in mammals, including cattle. In the present study, we identified novel and differentially expressed miRNAs in the testis and ovary in Holstein cattle by combining the Solexa sequencing with bioinformatics. In total 100 and 104 novel pre-miRNAs were identified in testicular and ovarian tissues, encoding 122 and 136 mature miRNAs, respectively. Of these, 6 miRNAs appear to be bovine-specific. A total of 246 known miRNAs were co-expressed in the testicular and ovarian tissues. Of the known miRNAs, twenty-one testis-specific and nine ovary-specific (1-23 reads) were found. Approximately 30.5% of the known bovine miRNAs in this study were found to have >2-fold differential expression within the two respective reproductive organ systems. The putative miRNA target genes of miRNAs were involved in pathways associated with reproductive physiology. Both known and novel tissue-specific miRNAs are expressed by Real-time quantitative PCR analysis in dairy cattle. This study expands the number of miRNAs known to be expressed in cattle. The patterns of miRNAs expression differed significantly between the bovine testicular and ovarian tissues, which provide important information on sex differences in miRNA expression. Diverse miRNAs may play an important regulatory role in the development of the reproductive organs in Holstein cattle.