Takane et al. BMC Genomics 2010, 11:101 http://www.biomedcentral.com/1471-2164/11/101 Page 9 of 13 Figure 4 Example of the 3’-UTR reporter plasmid and experimental validation. The 3’-UTR sequences of DLL1, CNN3, LARP4, ELK3, EIF2C4, TAGLN2, and ATP6V1B2 were subcloned into the XhoI/NotI site of the psiCHECK™-2 vector. CNN3 was chosen as representative of the eight candidates listed above (see Methods section). (B) HeLa cells were cotransfected with each combination of 100 ng of reporter plasmid and the indicated amounts of each miRNA (DLL1, 5 pmol of miR-34 (a); CNN3, 60 pmol of miR-1 (b); LARP4, 20 pmol of miR-1 (c); ELK3, 60 pmol of miR- 124 (d); EIF2C4, 60 pmol of let-7 (e), TAGLN2, 5 pmol of miR-1 (f), ATP6V1V2, 60 pmol of miR-1 (g), and LARP4, 60 pmol of let-7 (h)). Colours depict each miRNA: miR-34 (red), miR-1 (blue), miR-124 (orange), let-7 (green), and negative control (black). The relative expression of the luciferase gene was measured 24 h after transfection. The normalized luciferase activity of the control vector was set as 1.0. The data represent the average of three experiments and SDs. * P < 0.01. the nervous system [9]. Similarly, one of the evolutiona- rily conserved target candidates, the glucosamine-fruc- tose-6-phosphate aminotransferase 2 (GFPT2) gene, is expressed in the central nervous system [37]. In addi- tion, the annexin A7 (ANXA7) gene, which is another candidate target of miR-124, is involved in the develop- ment of the murine brain [38]. The striking overlap between the tissue specificity of evolutionarily conserved miRNA and that of their target genes suggests that one of the main functions of primordial miRNAs may have been the regulation of genes implicated in the tempor- ary control of the development of muscle and of the nervous system, in a tissue-specific manner. Finally, we found two interesting candidate genes, EIF2C4 and LARP4, which encode translation-related proteins. It is well accepted that miRNAs are regulators of gene expression, mostly at the translational level [1]. EIF2C4 is also known as Argonaute 4 (AGO4). Although the function of AGO4 is unknown, other Argonaute protein family members are involved in the RNA- induced silencing complex (RISC), which is essential for the miRNA or siRNA pathways. A previous microarray analysis performed in HepG2 cells revealed that the EIF2C4 gene was affected by let-7 [6]. In the present study, we demonstrated for the first time the direct downregulation of EIF2C4 by let-7, as assessed using a reporter gene assay in HeLa cells (Figure 4B, e); there- fore, we speculate that negative-feedback regulation of EIF2C4 by let-7 exists in the miRNA pathway. It has been reported that regulation of the AGO1 mRNA, which is a major component of the RISC complex, in the miRNA pathway by miR-168 controls plant develop- ment in Arabidopsis thaliana [39]. Another candidate, LARP4, encodes a member of La-motif protein family that controls translational efficiency [40]. We also demonstrated the downregulation of the LARP4 gene via miR-1 using a reporter gene assay (Figure 4B, c), which further supports our contention that some of the evolutionarily conserved miRNAs may play an important role in the regulation of translation by controlling the expression levels of translation factors and by negatively regulating their own miRNA pathway. Conclusions We developed a procedure to extract potential evolutio- narily conserved miRNA/target-gene pairs based on orthologous gene information from five bilaterian ani- mals, and efficiently extracted 31 evolutionarily