4.1 Introduction
Before starting this section, we should note that this part was also done by our co-group member, Feng-Mao Linand Chih-Hung Chou.
MicroRNA, a small non-coding RNA sequence (18~25 nt), plays important roles in gene regulations (7). They regulate gene expressions via recognizing the binding site in three prime untranslated region (3’UTR). Currently, many miRNAs and their target genes have been identified in a wide variety of species (79,149-151). The computational and experiment studies of miRNA target sites show that the sites are usually located in the conserved 3’UTR region of the orthologous genes (96,152). Since the first miRNA is discovered from Caenorhabditis elegans in 1993 (153), around 15,000 mature miRNAs have been discovered and stored in miRBase (150) and around 3,500 experimental validated miRNA-target interactions have been collected in miRTarbase (149). In order to understand how miRNA suppresses gene expression, many miRNA target site prediction tools were also proposed such as TargetScan (96), RNAHybrid (70), miRanda (138), PITA(85), PicTar (84) and so on. The target site prediction tools based on sequence complementary usually provide many false positive target sites. In order to reduce the false positive sites, the miRNA target sites located at the conserved sequences among various groups of organisms have higher potential to be the functional sites (96,99).
To extent the concept of finding miRNA target sites in conserved sequences among multiple orthologous species. A database, HomoloMTIs, were presented which integrated the resources of experimental validated miRNA target sites, predicted miRNA target sites based on the homologous genes from the HomoloGene. The predicted target sites having experimental validated miRNA target sites in homologous genes were annotated to strengthen the possibility the homologous species might have similar miRNAs or miRNA target sites. This database might reveal novel target sites or miRNA among various species.
4.2 Specific aims
MicroRNA plays important roles in post-translational gene regulation among various species. Nowadays many miRNA target interaction are revealed by experiments and miRNA target site prediction tools. In order to provide more evidences for predicted miRNA target interactions and discover real miRNA target interactions, a database, HomoloMTIs, was contructed based on three databases, miRTarBase, miRBase and HomoloGene, to reveals the miRNA target interactions might be shared in homologous genes. The homologous MTIs profiles could reveal the homologous genes regulated by miRNA with at least one experimental validation in one of the homologous genes and predicted miRNA target interactions. The novel miRNA target interactions and miRNAs could be pioneer studied by HomoloMTIs. HomoloMTI aims to provide a comprehensive comparative perspective on the metazoan repertoire of miRNA-target interactions as complementary to miRTarBase, the database of experimentally verified miRNA-target interactions.
4.3 Materials and method
Three databases are required for producing homologous miRNA-target interaction (MTI) database. The experimental validated MTIs were obtained from miRTarBase release 1.0 including 3,576 MTIs. The homologous genes were obtained from HomoloGene release 64. The miRNA sequences were obtained from miRBase release 14.
Each profile of homologous MTI was constructed based on one of the experimental validated MTIs in miRTarBase. Figure 25 illustrates the construction of the homologous MTI profiles. Each experimental validated MTI contains one miRNA and one target gene.
The homologous genes of the target gene from experimental validated MTI were collected from HomoloGene. According the miRNA in experimental validated MTIs, miRNAs having identical suffix was collected. Then, the miRNA target sites of
Figure 25. The construction diagram of HomoloMTIs. The database integrated three databases and predicted miRNA target sites on the homologous genes genes based on the experimental proved MTIs from miRTarBase.
4.4 Results
4.4.1 Statistics
There are 1,658 miRNA-target interactions between 430 miRNAs and 1,006 target genes validated by luciferase reporter assay AND western blot. To obtain the corresponding homologous gene pairs, we required the related metazoan genes in NCBI HomoloGene. Table 10 lists the number of homologous MTIs in each species. The overall homologous MTI groups is 1,569 which means some groups were shared in many species. For example, human HomoloMTIs includes 229 miRNAs and 618 target genes extracted from 1,114 MTIs in miRTarBase. Although, the MTIs of Chimpanzee, Cattle and Dog cannot be found in miRTarBase, some homologous MTI groups included their homologous genes.
Table 10. Statistics of homologous miRNA-target interactions collected in HomoloMTI
Species
No. of miRNA-target
interactions No. of miRNAs in
miRTarBase No. of target genes in miRTarBase miRTarBase
MTIs
HomoloMTI groups
Human 1114 1411 229 618
Chimpanzee - 1017 - -
Mouse 301 1356 107 222
Rat 46 671 31 29
Chicken 12 647 5 12
Cattle - 769 - -
Dog - 544 - -
Zebrafish 73 657 23 54
Fruit fly 96 234 28 58
Nematode 16 78 7 13
Total 1658 1569 430 1006
HomoloMTI aims to provide a comprehensive comparative perspective on the metazoan repertoire of miRNA-target interactions as complementary to miRTarBase, the database of experimentally verified miRNA-target interactions.
4.4.2 Case Study of homologous miR-122::ALDOA
Experimentally verified MTIs included in HomoloMTI was retrieved from miRTarBase. A homologous MTI group was constructed based on the experimental validated MTI. For example, HMTI000328 shown in Figure 26 was created from an experimental validated MTI in human and mouse. The homologous MTIs of cattle, rat, zebrafish and dog were predicted with their own miR-122 and homologous genes of ALDOA. The MTI in the species without miR-122 are predicted based on validated interactions of miR-122.
Figure 26. The homologous MTI group of miR-122 and target gene ALDOA.