Description of the bovine ovarian small RNA library
To identify miRNAs in the ovary, RNAs of 18 to 26 nt in length from bovine ovarian small RNAs (~200 nt) were purified, cloned, sequenced and analyzed. About 233 concatemer clones were sequenced to generate 479 sequences (after discarding non-quality and self ligated linker sequences). Of these 80 small RNA-cDNA sequences were beyond the expected range of nucleotides (18-26nt) in length. Only sequences of 18 nt or more in length were subjected to detail analysis. Distribution of different lengths of nucleotide sequences found in this library is presented in figure . We categorized all identified sequences according to their properties as determined by in-silico analysis based on the criteria reported elsewhere for different types of small RNAs [26
]. The 479 sequences identified in the library represented 41% miRNAs, 12% mRNA, 12% rRNA, 6.3% tRNA, 6.0% repeat associated siRNA, 2.7% small antisense RNA, 3.5% tiny noncoding RNA, 1% small nuclear RNA and 15.2% sequences that did not match to bovine genome (Figure ).
Size distribution of 479 small RNAs sequences cloned from the bovine ovary.
Frequency (%) of different types of RNA represented in the library.
Distinct miRNAs identified in the bovine ovary
In cDNA library a total of 196 sequences were found to be miRNA like molecules, of which 74 revealed distinct miRNAs (Table , Additional file 1
). Out of these 74 miRNAs, 36 were found to be reported in miRBase 12.0 for different species including bovine, 14 are registered only for other species and 24 were completely new. Of these 38 new bovine miRNAs, 15 miRNAs were identical or differed by only one or two nucleotides from known mammalian miRNAs. We denoted all the new miRNAs starting with prefix 'bomir' followed by their homologue miRNA number or by clone name in case of no sequence homology. Already annotated miRNAs were named as they were stated in miRBase.
List of new miRNAs cloned from bovine ovary
Two miRNAs, namely: mir-22/22* and 140/140* which are cloned from 5' fold back arm of the hairpin precursor, have shown exact match to human miRNAs but not to bovine as annotated in miRBase. So, previously annotated bta-miR-22 and 140 seem to be miR-22* and miR-140*, respectively. The number of times that each miRNA cloned in the library ranged from 28 clones for let-7b to a single clone (singleton) for 39 of the 73 miRNAs. All in all, 22 of the 73 miRNAs were cloned for three or more times (Figure , Additional file 1
Frequency (%) of cloned miRNAs along with their copy number.
The corresponding bovine genomic sequences and their locations were identified for each miRNA. The 5' or 3' flanking genomic sequences were then tested for the ability to fold into canonical ~70-nt miRNA precursor hairpin structures by using the MFOLD web server [34
]. Small RNA clones with proper positioning within an arm of the hairpin suggest that they have been excised during dicer processing in the cells. Nearly in all of those cases, sequences were found to be conserved in different species including the predicted precursors (Additional file 2
). The Bomir-652, which could not be located in bovine genome, was found to be cloned for five times in the library and share sequence homology with already identified miRNA in other species.
Genomic distribution, properties and clustering of new miRNAs
Genomic locations and properties of the new miRNAs are shown in table . All newly identified bovine miRNAs (except bomir-652) are corresponded to 43 distinct loci. Putative precursor hairpin structures have been predicted for all these 43 loci using genomic sequences flanked from candidate miRNAs (Additional file 2
). Thirty three of these are found to be encoded by single copy miRNA genes, whereas the other five (bomir-378, bomir-C0533-5p, bomir-F0522-3p, bomir-A3341-3p and bomir-A4052-5p) have multiple loci in the bovine genome (Additional file 2
). The analysis of the genomic positions of 61 sequences corresponding to 38 distinct new miRNA genes showed that the majority (23 out of 44 loci) are localized to intergenic regions and the rest corresponded to the intragenic regions in either sense or antisense orientation (Additional file 2
). However, 11 sequences are found to be exclusively from known intronic region.
Characterization of our miRNAs was done based on the annotation in the bovine genome data base Ensembl 52: Btau_4.0 [35
]. Bomir-F0522-3p and bomir-A4052-5p were mapped to both intergenic and intronic locations. Bomir-F0132-5p (sense), bomir-E2664-3p (antisense) and bomir-A4052-5p (antisense) are originated from the exons of protein-coding genes. While searching the genomic location for all miRNAs, we found six new genomic locations for annotated miRNAs like bta-mir-106, 24, 26, 199a and let-7b (Additional file 1
All the 50 new genomic loci were found to be distributed in 19 chromosomes (Chr.) namely: Chr. 3, 4, 5, 7, 8, 9, 11, 12, 14, 16, 18, 19, 20, 21, 22, 25, 26, 28 and 29. However, eight loci were found to be mapped to unknown chromosome in the Ensembl 52: Btau_4.0 (end note). Among all newly identified loci, eight miRNA genes were found to be located on Chr. 18 and five miRNAs found on Chr. 7 and 21. Further analysis of the already annotated miRNAs and the newly predicted loci has revealed six miRNAs gene clusters which were mapped within < 10 kb. This clusters are i) bta-miR-10a and bomiR-A0321 on Chr. 19; ii) bta-miR-23b, bta-miR-27b and bta-miR-24-3 on Chr. 8; iii) bta-let-7a-3 and let 7b-2-3P on Chr. 5; iv) bomiR-A4052-1 and bomiR-C0533 on Chr. 18; v) bta-miR-487a, bta-miR-487-b, bomiR-382 and bomiR-409 on Chr. 21; vi) bomiR-C0533-2 and bomiR-A4052-2 on Chr. 21.
To determine whether our new miRNAs are conserved among closely related species, we have searched for homology for precursor sequence in the ENSEMBL genome databases. Results revealed that 17 precursor loci (out of 43 loci for 38 new bovine miRNAs) were found to be conserved in at least six species. While five miRNAs (bomiR-F0244, bomiR-A0321, bomiR-F2531, bomiR-D3011 and bomiR-A3711) were found to be conserved in at least two species, 21 miRNA loci were specific to bovine. All of the newly cloned miRNAs were found to be conserved as mature sequences in the genome of different species. Thermo-dynamically stable hairpin structures were found for those conserved and new miRNAs as shown in additional file 2
Other small RNAs and their genomic properties found in the library
Analysis of small RNA library in the present study has enabled us to identify 57 different endogenous siRNAs. We categorized them broadly into two groups, namely: 29 sequences composed of 27 distinct RNAs derived from genomic repetitive region as repeat associated small interfering RNAs (rasiRNAs) and other 30 RNAs associated to non repetitive regions as non-repeat associated small interfering RNAs (nasiRNAs). According to their sequence properties 13 out of 30 nasiRNAs were found to be natural antisense transcripts with ~20 nt in length. Therefore, since they seem to be endogenous siRNAs, we denoted them as small antisense RNAs (santRNAs) and the rest 17 as tiny non-coding RNAs (tncRNAs). Size ranges for rasiRNAs were 18-28 nt (with mean ± SD 21.5 ± 3.1 nt), which did not revealed a sharp size distribution characteristic. However, for the santRNAs and tncRNAs the size distribution was 19.6 ± 1.9 and 19.5 ± 1.1 nt, respectively. Cloned rasiRNAs were found to be distributed on various chromosomes and mapped to repeat sequences mostly corresponding to retrotransposons in both sense and antisense orientation. Total numbers of hits for 27 rasiRNAs were 581 (ranging from 4 to100). Seventy five percent of the rasiRNAs were found to have preference for uridine and adenine residues in either 3' or 5' end position. While seven of the santRNAs were precisely mapped to intergenic region, six fitted to intronic region. All the 13 santRNAs were cloned as antisense orientation to the genome or intron of the protein coding genes on 12 different chromosomes.
Secondary structure analysis of all santRNAs revealed no characteristic hairpin as found for the miRNAs. While eleven tncRNAs were mapped to intergenic region, five were mapped to intronic and two to exonic regions. Two of the seventeen tncRNAs were predicted to form potential fold back structures like the miRNAs. However, these putative tncRNA precursor structures deviated significantly from the miRNA hairpins in key features and they were found to be poorly conserved in closely related species.
Detection and expression of miRNAs in the ovary and other bovine tissues
The expression of all new miRNAs including nine annotated miRNAs (let-7b, mir-15b, mir-18a, mir-29a, mir-125b, mir-126, mir-145, mir-199a and mir-222) in 11 different bovine tissues were analyzed using semi-quantitative RT-PCR (details in Figure , Table and Additional file 2
). As small RNAs were cloned in the library derived from all compartments of the ovary, samples from ovarian cortex, cumulus cells and matured corpus luteum were used to determine the sub-cellular expression profile of the new miRNA using RT-PCR (Table ). This is because of two facts: firstly, the bovine ovary is continuously changing throughout the process of folliculogenesis and secondly, the distinct nature of function of intra-ovarian cells and tissues compartments in the ovary.
Figure 4 Detection and expression analysis of selected miRNAs in multiple tissues. Expression profiles of some representative miRNAs (out of detected 44 miRNAs) in multiple tissues by PCR approach. While the figures are presented in the additional file 2 and the (more ...)
Detection and expression of selected miRNAs in multiple tissues
Of all 47 miRNAs (38 new and 9 already annotated miRNAs) 44 were detected in both ovarian cells and multiple tissues. Five miRNAs (bta-mir-126, bomir-F0132, bomir-A0321 and bomir-F1821) were found to be expressed at similar level in all experimental tissues. Seven miRNAs (bta-mir-18a, bta-mir-29a, bomir-140, bta-mir-199, bomir-378, bomir-F0132 and bomir-F2422) were found to be expressed at relatively higher levels in ovarian cortical portion (Table ). On the other hand, all undetected or less expressed miRNAs in ovarian cortex were found to be highly expressed in cumulus cells or corpus luteum. Most of the miRNAs were found to be differentially expressed between adult ovarian tissues and fetal ovary. Among them bta-mir-15b, bomir-409, bomir-652, bomir-C0533 and bomir-D1431 were highly expressed in the fetal ovary compared to that of adult ovarian cortex. However, bta-mir-29a, bta-mir-199 and bomir-F2422 were found to be expressed at higher level in the adult ovarian cortex than that of the fetal ovary (Table ). Bta-mir-125b, bta-mir-222, bomir-542, bomir-652, bomir-H0222, bomir-F0522, bomir-C1931 and bomir-A2143 were found to be expressed at very low level or not detected at all in the ovarian cortex. However, their abundance was higher in the cumulus cells and matured corpus luteum. The expression of bta-mir-222 was detected exclusively in the cumulus cells. In addition, higher expression of bta-mir-125b, bomir-409, bomir-503 and bomir-F0244 was also observed in the cumulus cells. The expression of bomir-652, bomir-H0222, bomir-C1931 and bomir-A2143 was higher in the corpus luteum.
Moreover, higher expression level of different miRNAs in various reproductive tissues was also observed. This includes bomir-940 in the oviduct; bta-mir-222, bomir-F2422 and bomir-G2511 in the uterus; and bta-mir-29a, bomir-143, bta-mir-145, bta-mir-199, bomir-542 in the placenta. All these investigated miRNAs were detected at least in one of the non-ovarian somatic tissues including heart, liver, lung and spleen (Table ). The RT-PCR analysis did not confirm the expression of three novel miRNAs (bomir-F0131, bomir-H0121 and bomir-B0821) in any of the tissues under investigation (image not shown).
In order to elucidate the cellular localization of one miRNA, bta-miR-29a was selected due to its differential expression between adult and fetal ovary, which are distinct in their functional activity. Accordingly, in-situ localization of this miRNA in the sections of bovine ovarian follicle revealed its expression in the different intra-ovarian cells (theca, mural granulosa, cumulus granulosa and oocyte) of different stages of development including primordial, primary, growing and matured/tertiary follicles (Figure ). Stable expression was detected in the whole mount cumulus-oocyte-complexes derived from the follicles of more than 8 mm in diameter. In the semi-quantitative RT-PCR data, expression of this miRNA was found in the cortex region of the adult ovary where follicles with cumulus cells are residing. Moreover, the expression of this miRNA was detected further until early stage of corpus luteum (Figure ), but very low or no expression in the matured corpus luteum (Figure ).
Figure 5 In-situ detection of mir-29a in the ovarian sections and whole mount COCs. Bovine ovarian cryo-sections and whole mount cumulus-oocyte complexes were in situ hybridized with 3'-digoxigenin labeled locked nucleic acid (LNA) microRNA probes for miR-29a (more ...)
Prediction and functional categorization of cloned miRNA targets
The goal of this prediction and analysis was to find the major biological processes and signaling pathways in the ovary that are most likely affected by a group of miRNAs. Even though there were many potential target genes predicted for the cloned miRNAs, several filtering and screening procedures (see materials and methods) have enabled us to generate a comprehensive target list consisting of 115 potential genes from all the predicted targets (Additional file 3
). From this screened target set, we found that let-7b, mir-15b, mir-18a, mir-29a, mir-101, mir-125b, mir-126, mir-143, mir-145, mir-199a and mir-222 to have the highest number and overlapping targets (Figure ). Interestingly, we found that all of these targeting miRNAs were represented at higher frequency in our constructed library.
Figure 6 Eleven miRNAs with highest number of screened target genes (sub-set miRNAs targets). Each circle representing one miRNAs and the surrounding genes are targeted by that miRNA. Genes shared by the different circles highlighted as blue (overlapping genes (more ...)
Detailed Gene Ontology (GO) analysis of the screened and sub-sets of miRNAs target genes were found to be associated with reproductive system development, function and disorders. These include cell cycle, morphology, cell death, cell to cell signaling, cellular growth, development and proliferation, DNA replication, recombination & repair, endocrine system disorder and different pathways underlying the ovarian functions. To further elucidate the specific functions of these genes, a detailed pathway analysis was performed using Ingenuity Pathway Analysis (Redwood City, California) for all target sets (Figure ) as well as for the sub-set of genes (Table , Additional file 3
Figure 7 Top biological function, disease categories and pathways enriched with predicted and screened miRNA target genes. A. Top biological functions and disease categories and B. pathways enriched with predicted and screened miRNA target genes. Ratio is the (more ...)
Ingenuity analysis of the genes targeted by top eleven screened miRNAs