Characterization of bovine miRNAs by sequencing and bioinformatics analysis
© Jin et al; licensee BioMed Central Ltd. 2009
Received: 07 May 2009
Accepted: 16 September 2009
Published: 16 September 2009
MicroRNAs (miRNAs) are a family of ~22 nucleotide small RNA molecules which regulate gene expression by fully or partially binding to their complementary sequences in mRNAs or promoters. A large number of miRNAs and their expression patterns have been reported in human, mouse and rat. However, miRNAs and their expression patterns in live stock species such as beef cattle are not well studied.
We constructed and sequenced small-RNA libraries to yield a total of 13,541 small-RNA sequences from 11 bovine tissues including brain, subcutaneous fat, muscle, liver, kidney, spleen and thymus. In total, 228 miRNAs including 29 novel miRNA candidates were identified. Of the 199 miRNAs, 101 have been previously reported as bovine miRNAs and the other 98 are bovine orthologs of known miRNAs that have been identified in at least one other mammalian species. Of the 29 novel miRNA candidates, 17 appeared at this point in time to be bovine specific, while the remaining 12 had evidence of evolutionary conservation in other mammalian species. Five miRNAs (miR-23a, -23b, -99a, -125b and -126-5p) were very abundant across the 11 tissues, accounting for 44.3% of all small RNA sequences. The expression analysis of selected miRNAs using qRT-PCR also showed that miR-26a and -99a were highly expressed in all tissues, while miR-122 and miR-133a were predominantly expressed in liver and muscle, respectively.
The miRNA expression patterns among 11 tissues from beef cattle revealed that most miRNAs were ubiquitously expressed in all tissues, while only a few miRNAs were tissue specific. Only 60% miRNAs in this study were found to display strand bias, suggesting that there are some key factors for mature miRNA selection other than internal stability. Most bovine miRNAs are highly conserved in other three mammalian species, indicating that these miRNAs may have a role in different species that are potential molecular markers for evolution.
MicroRNAs (miRNAs) are small non-coding RNA molecules of approximately 22 nucleotides in length, which play important regulatory roles in animals and plants [1, 2]. miRNAs have been found to down-regulate the expression of target genes by binding to the complementary sites in transcripts and causing translational repression or transcript degradation . Numerous biological processes in animal development, apoptosis, fat metabolism and hematopoietic differentiation have been reported to be regulated by miRNAs [4–7]. In addition, recent studies have revealed that miRNAs can increase protein translation by binding to complementary promoter sequences, extending the important function of miRNA to protein expression [8, 9].
Many experimental techniques and computational methods have been developed to identify miRNAs [10–12]; and a large number of miRNAs have been identified in primates, rodents, birds, fish and plants [13–16]. However, the number of miRNAs from bovine species is limited with only 125 reported (miRBase 12.0, December 2008), comparing with 866 from human and 627 from mouse. Early studies suggested that most miRNAs are conserved among related species [17, 18]. However, recent studies have shown that many newly identified miRNAs are species specific , and the expression of miRNAs is not strictly conserved among species . Although many miRNAs are known to be differentially expressed during development and across tissue types [4–6, 21], little is known about the relative abundance and specificity of expression patterns among tissues for most bovine miRNAs. In this study, we profiled bovine miRNAs and evaluated their expression patterns from 11 beef cattle tissues including muscle, kidney, liver, spleen, thymus, three fat tissues and three brain tissues. Elucidation of the expression patterns of different miRNAs among different tissues will contribute to the understanding of the roles of miRNAs in gene expression regulatory networks for particular biological functions in livestock species.
Identification of miRNAs and novel miRNA candidates
An overview of sequencing results from 11 small RNA libraries
Sequences of reported bovine miRNAs
Sequences of orthologs of known miRNAs
Sequences of novel miRNA candidates
Sequences of other small RNAs
Reported bovine miRNAs
Orthologs of known miRNAs
Novel miRNA candidates
Novel miRNA candidates identified in this study
H; C; E
H; M; R; C; E
H; M; R; C; E
H; C; E
H; C; E
H; C; E
H; M; R; C; E
H; M; R; C; E; G
H; M; R; C
To investigate evolutionary conservation of these miRNA candidates, six vertebrate genomes including human, mouse, rat, dog, horse and chicken were taken into account. Twelve out of 29 miRNA candidates were found to be conserved in at least one of these species. Three miRNA candidates (bta-un10, bta-un13 and bta-un20) were identified in the 5' arm of bta-mir-381, -495 and -487a, respectively, while only the 3' arms of these miRNAs were reported in several species (Table 2, Additional file 4).
A total of 207 genes encoding the identified bovine miRNAs were predicted using reported bovine miRNAs from miRBase 12.0 and the identified bovine orthologs of other mammalian miRNAs. Of these, 117 were reported in miRBase (version 12.0); 19 miRNA precursors were not reported in miRBase database previously; 71 were detected based on the orthologous miRNAs (Additional file 2). More than 30 bovine miRNAs were found to be encoded from more than one predicted hairpin precursor.
When the bovine miRNAs were compared to those from human, mouse and rat, most bovine mature miRNAs were also reported in the other three mammalian species. This agreed with that many miRNAs are conserved [17, 18] among mammalian species. Most of the conserved miRNAs in the four species were also found to have the same number of precursor(s) although they were encoded by multi-copies of genes (For example, let-7a as shown in Additional file 5), indicating that the processing of these miRNAs has been conserved among these species.
Sequence variations and end variants in mature miRNA sequences
It has been reported that a variant of end (size) different siRNAs or miRNAs could be generated during Dicer processing of double-stranded RNA (dsRNA), short hairpin RNA (shRNA) and miRNA precursors in vitro [24, 25]. Alignment of the identified sequences revealed that end variants were present in most miRNAs, showing that many miRNAs sequenced more than two times had end variants that were apparently generated from the same precursor (Additional file 3). Most end variants differed at the 3' end nucleotide(s) and a small percentage of them differed at the 5' end. The finding of the variant miRNA sequences is consistent with previous large scale miRNA profiling studies [13, 14]. More than 10 bovine miRNAs reported in miRBase were one or two nucleotides shorter at the 3' end comparing to ours, for example, bta-miR-18a and bta-miR-16b (Additional files 2 &3).
Analysis of miRNA expression in bovine tissues
MiRNA expression patterns in different tissues have been profiled in several vertebrates [13, 26–30]. Although there were diverse expression profiles in different tissues, most miRNAs were ubiquitously expressed. Our comparison of miRNA expression across 11 tissues from bovine revealed a few tissue specific miRNAs: miR-9, -124 in brain, miR-122 in liver, miR-1, miR-133a and -206 in muscle, which had been previously reported in mouse and human [13, 27]. Brain and muscle tissues have much more specific or enriched miRNAs than other tissues especially fat tissues, indicating that these miRNAs may play important regulatory roles in these tissues.
MiRNAs may be gained or lost during evolution, however, many miRNAs are conserved [17, 18]. Identification of common miRNA precursors between bovine and other three mammalian species (human, mice, and rat) suggests these miRNAs may have similar roles as those in other species since these conserved miRNA have higher expression levels [14, 31]. The higher expression level of three conserved miRNAs (miR-26a, -99a and -150) in all tested bovine tissues suggest that these miRNA may be more relevant to the highly conserved biological process in mammalians. Further study to discover their regulatory functions are needed. Recent developed hhigh-throughput sequencing analysis has allowed the identification of an increasing number of species-specific miRNAs [12, 32] since these miRNA may play a role in host-specific biological process. Most of the miRNA candidates identified in this study were bovine specific, although their expression was less than 1% comparing the total sequenced clones, suggesting further studies using deep-sequencing technologies [12, 14, 32] or specialized small RNA isolation and cloning procedures [19, 33] may help to identify and understand the functions of species specific miRNAs.
MiRNAs are firstly transcribed as pri-miRNAs and gave rise to short, 70-nucleotide stem-loop structures (pre-miRNAs) by the Drosha-DGCR8 complex [34, 35]. The hairpin structures are then processed by Dicer. During the process, both strands of a miRNA precursor can form functional miRNAs. However, most of the time only one arm becomes the mature miRNA or predominant miRNA, while the other degrades or generates star miRNA. Khvorova et al  suggested that the arm with lower thermodynamic stability at its 5' end becomes the mature miRNA. In this study, we identified 15 pairs of bovine miRNAs and star miRNAs. The stability of the initial four base pairs of these miRNA pairs were calculated using nearest-neighbour method and 2-state hybridization algorithm [37, 38]. Sixty percent of bovine miRNAs (9/15) displayed strand bias (Additional file 6). We also calculated the stability of human miRNAs and star miRNAs from miRBase 12.0 using the same method. In total, 175 pairs of miRNAs and star miRNAs were taken into account and 15 pairs of them were excluded from evaluation. Interestingly, only 60% (96/160) of human miRNAs exhibit strand bias, too (Additional file 7). We argue that there must be additional important factors other than internal stability to determine which arm of the miRNA precursor becomes the mature miRNA or miRNA* since the following observations can not be explained: (1) Most miRNAs observed in this study had a variant of isoforms generated by Dicer and a few of the 5' end variants even processed by Drosha (e.g., four bta-miR-23a variants had an additional A nucleotide at the 5' end, Additional file 3). (2) Some conserved pre-miRNAs express different mature miRNAs or star miRNAs depending on the species. For example, the stem-loop sequence of bta-miR-126 was perfectly matched to those from human, mouse and rat; however, in cattle and mouse, both strands were observed as mature miRNAs, while in human and rat, one strand generates miRNA and the other strand generates miRNA*. (3) Some mature miRNAs from the same precursors reverse to star miRNAs or vice versa in different tissues or development stage .
Our small-RNA cloning and sequencing approach was efficiently target mature miRNAs. However, to identify and understand the functions of bovine miRNAs, further studies using deep-sequencing technologies are required. The miRNA expression patterns among 11 tissues in beef cattle showed most miRNAs are ubiquitously expressed, suggesting that these miRNAs may play a role in a broad range of biological processes in various tissues. Although mature miRNAs have been reported to display strand bias, the mechanism which arm of pre-miRNA dominates still need further study. Most identified bovine miRNAs were found in other three mammalian species and are highly conserved during evolution, suggesting that these miRNAs may have similar function in mammalian species and can be potential molecular markers for evolution.
Tissues collection and RNA extraction
Bovine tissues including longissimus dorsi muscle, kidney, liver, spleen, thymus, cerebellum, medulla, hypothalamus, abdominal subcutaneous fat and rump subcutaneous fat were collected from two 16-moth-old female Angus cross-breed cattle at the University of Alberta farm, while back subcutaneous fat tissue was collected from two 16-moth-old male Charolais cattle. Tissues were immediately frozen in liquid nitrogen and stored at -80°C until use. Total RNA and highly enriched small RNA (<200 nt) from tissues were extracted using mirVana miRNA Isolation Kit (Ambion Inc, Austin, TX, USA) according to the manufacturer's instruction.
Small RNA library preparation and sequencing
Eleven cDNA libraries for small RNAs from the bovine tissues were constructed using miRCat™ Kit (IDT DNA Technologies, Coralville, IA, USA). Briefly, ~30 ug of enriched small RNA for each library was separated on 15% denatured polyacrylamide gels. The RNAs between 18 and 24 nt in size were recovered from the gels and ligated to the 3' linker using T4 RNA ligase. The ligated RNAs (~42 nt) were eluted from 15% denatured polyacrylamide gels and ligated to the 5' linker. The 5'-and 3' -linked RNAs (~62 nt) were reverse-transcribed to cDNAs. The cDNA products were amplified and the purified PCR products were digested by BanI and finally concatemerized and cloned into pCR2.1 TOPO vector using a TOPO cloning kit (Invitrogen, Carlsbad, CA, USA). The white colonies were randomly selected from the libraries and some of them were analyzed for inserts by PCR. The plasmids were extracted by MILLIPORE vacuum system and Montage Plasmid Miniprep Kit and sequenced by 3730 Sequencing Analyzer.
Small RNA sequence analysis
A bioinformatics pipeline was developed for miRNA analysis. The small RNA sequences 18-26 in size were extracted and mapped to the latest bovine genome assembly (ucsc_btau4). All sequences were searched for miRNA sequences in miRBase. Small RNA sequence that had less than two mismatches (or >90% identity) with mammalian miRNAs in miRBase 12.0 was considered as a homologous miRNA and sorted by name without species prefix. Homologous miRNAs were further divided into previously reported bovine miRNAs and bovine orthologs of known miRNAs. Other small RNA sequences (mRNA, rRNA, tRNA, snoRNA, piRNA and misc RNA) were filtered by using BLAST against the NCBI nr database http://www.ncbi.nlm.nih.gov/.
The genomic DNA sequences flanking the bovine miRNAs from miRBase 12.0, orthologs of known miRNAs and small RNA sequences mapped to the bovine genome but not aligned to any annotated RNA classes were obtained from the bovine genome assembly. Sequences were analyzed for hairpin structure using the UNAFold algorithm with default parameters or the Mfold web server at http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi. The hairpin structure containing putative miRNA was regarded as pre-miRNA if it contained neither large internal loops nor bulges, and at least 16 nt of the putative miRNA (if putative miRNA sequence length <22 nt, stretch 3' end to 22 nt) were paired with the other arm of the hairpin.
To investigate conservation of bovine novel miRNA candidates, highly similar DNA sequences corresponding to their precursors in the human, dog, mouse, rat, horse and chicken genome assemblies were found using BLATN NCBI GENOMES with word size = 7http://blast.ncbi.nlm.nih.gov/Blast.cgi. Sequence alignments covering > 70% identity and >75% of the length of precursors of bovine miRNA candidates were used for hairpin structure analyses as described above. The orthologs of bovine miRNA candidates were defined to be those who have proper hairpin structures and less than 3 nucleotides variation sequences with those in other species.
Calculation of thermodynamic stability
Bovine miRNAs were sorted by clone counts. If the clone count of one strand was 10 times more than that of the other strand, they were regarded as miRNA and miRNA*, respectively. Human star miRNAs and their correspondingly high-expressed miRNAs were downloaded from miRBase (version 12.0). We used two-state hybridization web sever to calculate the free energy at http://dinamelt.bioinfo.rpi.edu/twostate.php. The internal stability of the initial four bases of miRNA or miRNA* (5'→3') were calculated as the delta G or delta G* (Additional files Table S5 & S6).
Quantitative RT-PCR was performed using human TaqMan miRNA probes that had the same sequence as the bovine miRNAs. Reactions were performed following manufacturer's recommendations (Applied Biosystems, Foster City, CA, USA) except for 20 ng total RNA used.
Hierarchical clustering of miRNA expression was performed with PermutMatrix , using Pearson distance, average linkage and normalized columns (Z score). Clustering was performed only on miRNA sequenced at least 20 times. The relative cloning frequency for each miRNA was calculated as the number of sequences for each miRNA in a library divided by the total number of miRNA sequences for that library.
abdominal subcutaneous fat
back subcutaneous fat
rump subcutaneous fat
longissimus dorsi muscle
This work was supported by Alberta Livestock Industry Development Fund (2007F044R). The authors thank Mrs. Y. Meng for her technical report and Dr. U. Basu for her assistance in qRT-PCR analysis.
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