172 IDENTIFICATION OF MICRORNAS IN BOVINE OVARY

Tightly regulated expression and interaction of a multitude of genes for ovarian folliculogenesis leading to successful oocyte development could be regulated by recently identified new class of small RNAs of ~22 nt (i.e. microRNAs), which are already proved as one of the vital transcriptional regulators in different biological processes including development. But their presence and expression in bovine ovary has not yet been determined. Here, we have attempted to identify miRNAs in bovine ovary by small RNA-cDNA library construction through 5 ligation independent cloning. For this purpose, total RNA enriched with small RNA was isolated from ovary and size fractionated (18 to 24 nt) by denaturing PAGE. Extracted RNA was first 3′ linkered and after template switching by RT, the second 3′ linkering of the first strand cDNA was performed. These linkered small RNA-cDNAs were then amplified with linker-specific primers consisting of BAN I restriction sites, concatemerized by serial ligation, cloned into TOPO TA vector, and transformed into TOP 10 chemically competent cells. After screening, colonies were picked and sequenced. Bioinformatic analysis was done according to the published criteria for the small RNAs. From 233 clones a total of 479 reads were identified. Frequency of sequence length found in the library was 26.8% for ≤18 nt, 55.1% for 19 to 22 nt, and 18.1% for ≥23 nt. The total 479 sequences identified in the library represent 35% miRNAs, 12% mRNA, 12.1% rRNA, 5.6% tRNA, 4.2% repeat associated siRNA, 3.8% non-repeat-associated siRNA, 4% tiny noncoding RNA, 1% small nuclear RNA, and 16% sequences not matched to bovine genome. All 171 miR sequences comprised 79 distinct miRNAs, of which 45 miRNAs already annotated in miRBase for bovine and the other 34 miRNAs are new discoveries. Of the 34 newly identified miRNAs, 12 are described in other species but not yet in bovine. Most of the miRNAs cloned into multiple times, where let-7a cloned for 10, let-7b for 28, let-7c for 13, miR-21 for 4, miR-23b for 11, miR-24 for 7, miR-27a for 6, miR-126 for 4, and miR-143 for 11 times. Based on best hit score, P-value and free energy by online target prediction, some of the bta-miR identified in the library (let-7b, 15b, 18a, 23b, 101, 125b, 126, 140, 145, 199a) are found to target hundreds of genes related to follicular development, ovulation and hormonal regulation. Further functional characterization of some selected miRNAs including expression profiling and in situ localization in follicles of different size and cycles may supplement the results of this study and will enable us to gain insight into their relation to female fertility.