Takane et al. BMC Genomics 2010, 11:101 http://www.biomedcentral.com/1471-2164/11/101 conserved miRNA/target-gene pairs from 357,430 pairs. We experimentally validated the downregulation of six candidate pairs (out of six tested pairs) in HeLa cells, which suggests that our method using orthologous information was efficient in extracting evolutionarily conserved miRNA target-gene candidates. Our findings reveal that miRNA target sites were conserved among various species, and demonstrate that especially let-7-, miR-1-, and miR-124-mediated gene regulation may have played an important role throughout evolution, in processes such as development, differentiation, and mus- cle movement. Moreover, our results indicate that miRNA-mediated translational regulation as well as tis- sue-specific expression of miRNA/target-gene pairs may have already existed in the common bilaterian ancestor. In conclusion, our study will provide new insights into the early stages of miRNA function. Methods miRNA and 3’-UTR sequence data We downloaded 2,404 mature miRNA sequences (885 for H. sapiens, 689 for M. musculus, 520 for G. gallus, 153 for D. melanogaster, and 157 for C. elegans) from the miRBase, version 13.0 http://microrna.sanger.ac.uk/ sequences/[13]. We downloaded sequences correspond- ing to 3’-UTR using the Ensembl transcript ID annota- tion in FASTA format (40,498 transcripts for H. sapiens, 3,332 for M. musculus, 13,089 for G. gallus, 16,822 for D. melanogaster, and 13,560 for C. elegans) from the Ensmart database Ensembl release 53 http://www. ensembl.org/index.html[41]. Orthologous gene informa- tion was also downloaded from the Ensmart database Ensembl release 53. We obtained 145 experimentally verified miRNA/target-gene pairs from TarBase Version 5.0.1 http://diana.cslab.ece.ntua.gr/tarbase/[42]. Identification of miRNAs conserved among bilaterian animals The 2,404 miRNA sequences were aligned using ClustalX [43] with the following alignment parameters: gap opening, 22.50; gap extension, 0.83; and bootstrap value, 100. We checked the conservation of 2,404 miRNA sequences to extract evolutionarily conserved miRNAs. We defined the conservation threshold as an “overall sequence identity > 75% with complete matching of the seed sequence (1-7, 2-8, or 3-9 nucleotides from the miRNA’s 5’ end)"; furthermore, we introduced information on the phylogenetic relationship among miRNAs to extract reliably conserved miRNAs and used highly conserved miRNA families (category I) [24]. Extraction of evolutionarily conserved miRNA/target-gene pairs among bilaterian animals To extract evolutionarily conserved miRNA/target-gene pairs among bilaterian animals, we devised a three-step Page 10 of 13 filtering approach (Figure 1). In step 1, we predicted genes targeted by each of the five miRNAs (let-7, miR-1, miR-124, miR-125/lin-4, and miR-34) using RNAhybrid, which is fast and flexible software for miRNA target prediction, with the free-energy option and the seed- sequence option [15]. The RNA duplex free-energy filter was defined as the appropriate value that led to the effi- cient extraction of experimentally verified miRNA/tar- get-gene pairs. We also considered a complete match across the seed sequence (1-7, 2-8, or 3-9 nucleotides from the miRNA’s 5’ end), which was used as a filter by adding the seed option of RNAhybrid. In step 2, we used four binding parameters of the hybridization pattern of the miRNA/mRNA duplexes. According to a recent study, a binding rule is likely to exist for the recognition of target mRNAs by miRNAs [34]. Moreover, G-U wobble pairs within miRNA/ mRNA duplexes play a key role in the interaction with target mRNAs [44]. Subsequently, potential candidates were extracted using four binding parameters (number of mismatches of mRNA within the whole miRNA sequence, number of mismatches of miRNA within the whole miRNA sequence, number of G-U wobble pairs within the whole miRNA sequence, and number of G-U wobble pairs within the seed sequence) of the miRNA/ mRNA duplexes (Additional file 1). We used the hybri- dization pattern of experimentally verified and predicted miRNA/mRNA pairs to calculate coverage, by changing these binding parameters one by one (28-0 for the num- ber of mismatches of mRNA within the whole miRNA sequence; 14-0 for the number of mismatches of miRNA within the whole miRNA sequence; 10-0 for the number of G-U wobble pairs within the whole miRNA sequence; and 6-0 for the number of G-U wobble pairs within the seed sequence) (Figure 2A-D). The range of each of the four binding parameters was determined based on the coverage of experimentally verified and predicted miRNA/mRNA pairs. Parameter combinations were then plotted on a 2D graph by calculating the “ratio of experimentally verified miRNA/mRNA” and “Enrichment” using the points in the four parameter ranges (Figure 2E). The criterion “Enrichment” was defined and calculated as the value of “ratio of experi- mentally verified miRNA/target-gene pairs” divided by the “ratio of predicted miRNA/target-gene pairs”. We obtained the most effective combination of four binding parameters for extracting miRNA/target-gene pairs based on the EC value. Parameter conbination with highest EC value was selected. In step 3, orthologous gene information was used to extract orthologous genes targeted by the same type of miRNA. For the retrieval of evolutionarily conserved miRNA/target-gene pairs from various bilaterian ani- mals, we set the orthologous gene information criteria