Abstract:miRNA (micoRNA) is a very important post transcriptional regulatory factor, and have been identified in many species, whereas miRNA related studies were poor in horse (Equus caballus). In this study, we carried out small RNA sequencing on mixed sample of 8 tissues of Mongolian horse with Solexa sequencing technology and analysed expression abundance of conserved and candidate miRNA, and predicted their target genes with relevant bioinformatic analysis. 5 922 735 clean reads were obtained after trimming low quality reads and adapter sequences from 9 478 925 raw reads which were generated from Illumina Miseq platform. Among them, 5 199 802 reads (15~30 nt) were selected from clean reads for subsequent analysis. After length distribution analysis, we found that total sequences were abundant in 20~24 nt that mainly based on 22 nt, which similar with length distribution of unique sequences. For small RNA annotation, total sequences distributed most in known miRNAs (69%), while unique sequences distributed most in rRNA (49%). Then we compared unique sequences with known miRNAs in miRBase and identified 239 conserved miRNAs and 243 miRNA precursors. In addition, we predicted 510 miRNAs and 375 miRNA precursors with all sequences using Mireap software, in which 238 miRNAs could be matched with horse known miRNAs and 272 non-matched miRNAs might be horse candidate novel miRNAs. The minimal folding free-energy (MFEs) of miRNA precursors reached 18.2~62.8 kcal/mol, which accordant with the requirement of its secondary structural stability. 23 037 target genes and 1 974 153 target sites for all predicted miRNAs were predicted by using miRanda software. These results enrich the horse miRNA database and can provide useful information for further related researches.
Ambros V 2004. The functions of animal microRNAs[J]. Nature, 431(7006):350-355.Barrey E, Bonnamy B, Barrey E J, et al. 2010. Muscular microRNA expressions in healthy and myopathic horses suffering from polysaccharide storage myopathy or recurrent exertional rhabdomyolysis[J]. Equine veterinary journal. Supplement, (38):303-310.Bartel D P 2004. MicroRNAs: genomics, biogenesis, mechanism, and function[J]. Cell, 116(2):281-297.Bernstein E, Caudy A A, Hammond S M, et al. 2001. Role for a bidentate ribonuclease in the initiation step of RNA interference[J]. Nature, 409(6818):363-366.Bonnet E, Wuyts J, Rouze P, et al. 2004. Evidence that microRNA precursors, unlike other non-coding RNAs, have lower folding free energies than random sequences[J]. Bioinformatics, 20(17):2911-2917.Chowdhary B P, Paria N, Raudsepp T 2008. Potential applications of equine genomics in dissecting diseases and fertility[J]. Animal reproduction science, 107(3-4):208-218.Das P J, Mccarthy F, Vishnoi M, et al. 2013. Stallion sperm transcriptome comprises functionally coherent coding and regulatory RNAs as revealed by microarray analysis and RNA-seq[J]. PloS one, 8(2):e56535.Der J P, Barker M S, Wickett N J, et al. 2011. De novo characterization of the gametophyte transcriptome in bracken fern, Pteridium aquilinum[J]. BMC genomics, 12:99.Doench J G, Sharp P A 2004. Specificity of microRNA target selection in translational repression[J]. Genes & development, 18(5):504-511.Grimson A, Farh K K, Johnston W K, et al. 2007. MicroRNA targeting specificity in mammals: determinants beyond seed pairing[J]. Molecular cell, 27(1):91-105.Hammond S M, Bernstein E, Beach D, et al. 2000. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells[J]. Nature, 404(6775):293-296.Hwang H W, Mendell J T 2006. MicroRNAs in cell proliferation, cell death, and tumorigenesis[J]. British journal of cancer, 94(6):776-780.Jin W, Grant J R, Stothard P, et al. 2009. Characterization of bovine miRNAs by sequencing and bioinformatics analysis[J]. BMC molecular biology, 10:90.John B, Enright A J, Aravin A, et al. 2004. Human MicroRNA targets[J]. PLoS biology, 2(11):e363.Kim M C, Lee S W, Ryu D Y, et al. 2014. Identification and characterization of microRNAs in normal equine tissues by Next Generation Sequencing[J]. PloS one, 9(4):e93662.Kim V N 2005. Small RNAs: classification, biogenesis, and function[J]. Molecules and cells, 19(1):1-15.Kozomara A, Griffiths-Jones S 2011. miRBase: integrating microRNA annotation and deep-sequencing data[J]. Nucleic acids research, 39(Database issue):D152-157.Lacourt M, Gao C, Li A, et al. 2012. Relationship between cartilage and subchondral bone lesions in repetitive impact trauma-induced equine osteoarthritis[J]. Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society, 20(6):572-583.Lee R C, Ambros V 2001. An extensive class of small RNAs in Caenorhabditis elegans[J]. Science, 294(5543):862-864.Lee R C, Feinbaum R L, Ambros V 1993. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J]. Cell, 75(5):843-854.Li G, Li Y, Li X, et al. 2011. MicroRNA identity and abundance in developing swine adipose tissue as determined by Solexa sequencing[J]. Journal of cellular biochemistry, 112(5):1318-1328.Liu Q, Paroo Z 2010. Biochemical principles of small RNA pathways[J]. Annual review of biochemistry, 79:295-319.Llave C, Kasschau K D, Rector M A, et al. 2002. Endogenous and silencing-associated small RNAs in plants[J]. The Plant cell, 14(7):1605-1619.Mach N, Plancade S, Pacholewska A, et al. 2016. Integrated mRNA and miRNA expression profiling in blood reveals candidate biomarkers associated with endurance exercise in the horse[J]. Scientific reports, 6:22932.Millar A A, Waterhouse P M 2005. Plant and animal microRNAs: similarities and differences[J]. Functional & integrative genomics, 5(3):129-135.Reinhart B J, Slack F J, Basson M, et al. 2000. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans[J]. Nature, 403(6772):901-906.Reinhart B J, Weinstein E G, Rhoades M W, et al. 2002. MicroRNAs in plants[J]. Genes & development, 16(13):1616-1626.Shendure J, Ji H 2008. Next-generation DNA sequencing[J]. Nature biotechnology, 26(10):1135-1145.Song G, Wang L 2009. A conserved gene structure and expression regulation of miR-433 and miR-127 in mammals[J]. PloS one, 4(11):e7829.Stark A, Brennecke J, Russell R B, et al. 2003. Identification of Drosophila MicroRNA targets[J]. PLoS biology, 1(3):E60.Wade C M, Giulotto E, Sigurdsson S, et al. 2009. Genome sequence, comparative analysis, and population genetics of the domestic horse[J]. Science, 326(5954):865-867.Wang X J, Reyes J L, Chua N H, et al. 2004. Prediction and identification of Arabidopsis thaliana microRNAs and their mRNA targets[J]. Genome biology, 5(9):R65.Yekta S, Shih I H, Bartel D P 2004. MicroRNA-directed cleavage of HOXB8 mRNA[J]. Science, 304(5670):594-596.Zeng Y, Yi R, Cullen B R 2003. MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms[J]. Proceedings of the National Academy of Sciences of the United States of America, 100(17):9779-9784.Zhang B H, Pan X P, Cox S B, et al. 2006. Evidence that miRNAs are different from other RNAs[J]. Cellular and molecular life sciences : CMLS, 63(2):246-254.Zhang C 2009. Novel functions for small RNA molecules[J]. Current opinion in molecular therapeutics, 11(6):641-651.Zhou L, Li X, Liu Q, et al. 2011. Small RNA transcriptome investigation based on next-generation sequencing technology[J]. Journal of genetics and genomics = Yi chuan xue bao, 38(11):505-513.Zhou M, Wang Q, Sun J, et al. 2009. In silico detection and characteristics of novel microRNA genes in the Equus caballus genome using an integrated ab initio and comparative genomic approach[J]. Genomics, 94(2):125-131.