Computational Identification And Analysis Of MicroRNA Genes From Canis Familiaris

McroRNAs, as a family of non-coding small RNA 18-25nt in length, have beenprofoundly studied in recent years in plants, animals and virus. As an importantregulator in eukaryote organism, many study work had done to prove that microRNAhave abroad roles in the development of organism involving every aspect of cellphysical activity. The characterization of miRNA is still a hot academic domain forthe cause of the identified microRNA far from saturation. The scientists hadcompleted sequencing of dog genome, and through human/dog genomic pairalignment, it is found that dogs and humans have similar levels of overall nucleotidediversity, meanwhile, the species display extreme phenotypic diversity, therefore, allprovide a good flat for research disease related with human. More importantly, sixmiRNA were characterized in miRBase thus far. Above all, these provide a greatacademic and realistic meaning for idemifying miRNAs in dog genome.As for time- and tissue- speciality of miRNA expression and mis-leading ofdegradation of mRNA and other non-coding RNAs, there is limitation by usingexperimental RNomics. Therefore, we apply computational RNomics to predictmiRNA in dog based on sequence conservation of mature miRNAs and theirprecursor displaying hairpin structure, leading to identification of 357 novel miRNAs.The following is the summarization of our result:1. The loci of all 357 microRNA sequences ha genome lie in all 33 chromosomes ofgenome. Among these miRNAs, 281 sequences belong to single-copy in doggenome and the other 35 sequences belong to multi-copy. The number ofsequences in intergenic region is 222, in intron is 125, anti-sense to gene is 50,sense to gene is 75, and in exon is 6, in UTR is 3. All precursors ranged from 65to 120 nt in length, with average length of 80 nt.2. We have analyzed whether all miRNA could form stem-loop when joined withflanking sequence in genome through m-fold programs and the results showedthat a majority of miRNA have this property. Homologue of human miRNAs hadbeen validated by experiment, indicating that they can express stably. Meanwhile,8 novel miRNAs were found by searching EST database. Furthermore, we carryout RT-PCR assay of 5 novel miRNA, two of which can obtain the expected result,suggesting that they can express. 3. Trough analysis, we found that the 300 homologue of human miRNA belong to149 families, and miR-154, miR-17 and let-7 family have 15, 13 and 10,respectively. Most of families only have one members, the case is different fromplant. Members of animal family display three mode, identical, closely related orhomology.4. For conforming miRNAs are transcripted under common promoter, we analysis.these miRNAs using promoter analysis program, leading to identification of 142miRNAs grouping into 53 clusters controlled by promoter in upstream, amongwhich 28 clusters is made up of multi homologus miRNAs and 18 miRNA clusterform 8 distinct paralogous clusters. Further analysis indicate that some clusterhave multi-copies through duplication.5. Interestingly, we found a large miRNA cluster made up of 41 miRNAs, whichreside in imprinted region of chromosome 8 high conserved between human anddog, and another two clusters lie in upstream of this cluster, a miRNA clustercomposed of 5 miRNA and C/D snoRNA cluster.6. We found that 15 miRNA occur in repetitive region in genome, among which 7 liein LINE element and the others reside in SINE elements. Some of themcompletely reside in repetitive region, and some overlap with element. TwomiRNAs reside in intron of rRNA gene.7. A majority of miRNAs are conserved among animal through analysis. Most ofmiRNAs evolved by local tandem duplication and non-local duplication. Localtandem duplications result in paralogous sequences that are located on the sametranscript. Non-local duplications result in paralogous genes or gene clusters ondifferent chromosomes.8. We predict target of identified miRNAs using 3-UTR. Further analysis indicatethat a majority of target involved in almost all cellular processes, for example,development, metabolism, regulatory factor and signal transduction. The mostriched GO term is transcriptional factor. Meanwhile, some miRNAs might areinvolved in processes related with cancer. Most importantly, we identified targetgene unique to dog, which regulate special processes during dog development.Finally, we found that miR-186 can regulate expression of its host protein, resultin turnout of its protein. This is a self-regulatory feedback mechanism.