Takane et al. BMC Genomics 2010, 11:101 http://www.biomedcentral.com/1471-2164/11/101 Page 8 of 13 Figure 3 Two examples of miRNA target sites in orthologous gene transcripts. Potential target sites of miR-1 (blue arrows) in the 3’-UTR sequences of the orthologous CNN3 (A) and LARP4 (B) transcripts are shown. Predicted duplexes formed by the 3’-UTR sequences (top) and miRNAs (bottom) are shown in dotted boxes for each potential target site. The green bar on the H. sapiens 3’-UTR sequence indicates a DNA region used for the construction of the reporter plasmid pLuc-CNN3 (Figure 4A). See Additional file 3 for other candidates. (*) The length of the CNN3 3’-UTR is currently registered as a little shorter than that indicated (527 nt in size) and contains the miR-1 binding site (Ensembl release 53). Moreover, the semaphorin-6D precursor (SEMA6D) gene, plays an important role in cardiac morphogenesis during chick embryonic dev which was predicted as a candidate of miR-34 targeting, elopment [31]. The lin- 28 gene, which regulates developmental timing in C. ele- gans, is reportedly controlled by lin-4, as assessed using in vivo experiments [32]. Subsequently, the lin-28 gene was also found to be regulated by miR-125, which is an orthologous miRNA of lin-4, in H. sapiens and M. mus- culus [33]. Our prediction confirmed the regulation of orthologous lin-28 genes by lin-4/miR-125 miRNA in H. sapiens, M. musculus, and C. elegans and further sug- gested that a similar regulatory relationship was con- served in G. gallus. Interestingly, our prediction showed that the orthologous let-7/lin-28 pair was also evolutio- narily conserved among bilaterians. This feature had been validated experimentally in H. sapiens [34]; how- ever, in C. elegans, lin-28 is expressed in an early stage of development, while let-7 is expressed in a later stage of development, suggesting that regulation of lin-28 by let-7 is subtle in C. elegans [7]. It would be of interest to analyse the interaction of the let-7/lin-28 pair in other species, such as M. musculus and G. gallus, to understand whether stage-specific expression of let-7 was present in the common ancestor of bilaterian ani- mals or if it is a trait acquired later during evolution, as many of the evolutionarily conserved miRNA/target- gene pairs were related to an essential function involved in differentiation and development. Next, we focused on the tissue-specific miRNA/target genes. Among the 30 evolutionarily conserved target candidates, approximately one-third were expressed in a tissue-specific manner in humans, according to the BioGPS portal http://biogps.gnf.org. miR-1 is highly expressed in muscle tissues [8]. Here, three candidate genes regulated by miR-1 (i.e., TAGLN2, CNN3, and TPM3) are also expressed in muscle tissues, according to BioGPS. TAGLN2 is a homolog of TAGLN, which encodes an actin-binding protein and is a diagnostic marker of breast and colon carcinoma in humans [35]. The CNN3 gene also encodes an actin-binding protein that represses bone morphogenetic protein (BMP) sig- nalling in chondrocytes, which is important for bone formation [36]. Moreover, the TPM3 gene encodes yet another actin-binding protein that modulates muscle contraction. The other miRNA, miR-124, is expressed in