Characterization Of Micrornas From Sheep (ovis Aries) Using Computational And Experimental Analyses

MicroRNAs (miRNAs), about 22nt in length, are a class of non-coding small RNA distribute extensively in human genome and other eukaryote genome. They regulate gene expression through sequence specific interactions with target mRNAs resulting in translational repression or mRNA degradation. Recent findings showed that miRNAs play an important role in embryo formation, organogenesis, cell death, cell proliferation, and cancer development, and so on. By April 2010, the number of known miRNAs has grown rapidly to 14,197 in more than 130 species. O.aries (Ovis aries) is one of the most important agricultural livestock for meat, milk, and wool production. But there is limited information about O.aries miRNAs. Thus, to research on O.aries miRNAs is very important and necessary.In this study, we combined a computational method based on expressed sequence tag (EST) analysis with experimental identification based on small RNA cDNA library to identify O.aries miRNAs. Then, we analized their expression level in different sheep tissues to find out muscle specific miRNAs, predicted their target genes and conducted the function analysis for them. The results are as follows:1. First, by constructing four cDNA libraries for small RNAs in the size range of 18–26nt from the tissues of longissimus muscle, brain, liver and spleen, we cloned 17 O.aries miRNAs. Among these miRNAs, 12 were isolated from longissimus muscle, 6 were cloned from brain, and 2 others were cloned from liver and spleen.2. Second, we used a computational approach to exploit O.aries miRNAs in sheep EST database. Following a set of strict filtering criteria, we finally identified 14 O.aries miRNAs, which included 4 pairs of miRNA/miRNA*, and 1 pair of miRNA-3p/miRNA-5p. A miRNA cluster was also identified, including oar-miR-374b, oar-miR-374b* and oar-miR-421.3. Altogether, we identified 31 oar-miRNAs belong to 24 families, all of which were first identified in sheep. 2 of them were novel miRNAs that had never been annotated in miRBase database, named oar-NEW-1 and oar-NEW-2. The remaining 29 miRNAs were sheep orthologs of known mammalian miRNAs.4. Northern blot showed that 3 miRNAs appeared to be extremely tissue specific. oar-miR-1 could be only detected in heart muscle and longissimus muscle, oar-miR-122 could be only detected in liver, and oar-NEW-1 could be only detected in spleen.5. We chose 4 miRNAs (the pair of oar-miR-24/oar-miR-24-1* and the pair of oar-miR-425-3p/oar- miR-425-5p) identified by computational method for expression analysis by Real-time PCR. All of these four miRNAs could be detected in total tissues, except that oar-miR-425-5p could not be detected in heart muscle. The results also showed that oar-miR-24 had a significant higher expression level in longissimus muscle and heart muscle than which in the other tissues, as same as oar-miR-24-1* in heart muscle, oar-miR-425-3p in longissimus muscle, and oar-miR-425-5p in brain.6. Further, we predicted 120 potential target genes, included 230 target sites, for 31 oar-miRNAs in O.aries mRNAs. Gene ontology analysis showed that these target genes nearly took part in all kinds of biological process, and the molecular function which involved most is binding activity.