Takane et al. BMC Genomics 2010, 11:101 http://www.biomedcentral.com/1471-2164/11/101 R E S E A R C H A R T I C L E Open Access Computational prediction and experimental validation of evolutionarily conserved microRNA target genes in bilaterian animals Kahori Takane1,2, Kosuke Fujishima1,2, Yuka Watanabe1,2, Asako Sato1, Nobuto Saito1,2, Masaru Tomita1,2, Akio Kanai1,2* Abstract Background: In many eukaryotes, microRNAs (miRNAs) bind to complementary sites in the 3’-untranslated regions (3’-UTRs) of target messenger RNAs (mRNAs) and regulate their expression at the stage of translation. Recent studies have revealed that many miRNAs are evolutionarily conserved; however, the evolution of their target genes has yet to be systematically characterized. We sought to elucidate a set of conserved miRNA/target-gene pairs and to analyse the mechanism underlying miRNA-mediated gene regulation in the early stage of bilaterian evolution. Results: Initially, we extracted five evolutionarily conserved miRNAs (let-7, miR-1, miR-124, miR-125/lin-4, and miR-34) among five diverse bilaterian animals. Subsequently, we designed a procedure to predict evolutionarily conserved miRNA/target-gene pairs by introducing orthologous gene information. As a result, we extracted 31 orthologous miRNA/target-gene pairs that were conserved among at least four diverse bilaterian animals; the prediction set showed prominent enrichment of orthologous miRNA/target-gene pairs that were verified experimentally. Approximately 84% of the target genes were regulated by three miRNAs (let-7, miR-1, and miR-124) and their function was classified mainly into the following categories: development, muscle formation, cell adhesion, and gene regulation. We used a reporter gene assay to experimentally verify the downregulation of six candidate pairs (out of six tested pairs) in HeLa cells. Conclusions: The application of our new method enables the identification of 31 miRNA/target-gene pairs that were expected to have been regulated from the era of the common bilaterian ancestor. The downregulation of all six candidate pairs suggests that orthologous information contributed to the elucidation of the primordial set of genes that has been regulated by miRNAs; it was also an efficient tool for the elimination of false positives from the predicted candidates. In conclusion, our study identified potentially important miRNA-target pairs that were evolutionarily conserved throughout diverse bilaterian animals and that may provide new insights into early-stage miRNA functions. Background MicroRNAs (miRNAs) are a class of short (18-25 nucleo- tides) non-coding RNAs that regulate gene expression posttranscriptionally. Their regulatory potential relies heavily on the recognition of binding sites that are located mainly in the 3’-untranslated regions (3’-UTRs) of target messenger RNAs (mRNAs) [1]. Currently, numerous miR- NAs with diverse sequences are being characterized in a * Correspondence: akio@sfc.keio.ac.jp 1Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan wide range of species [2], suggesting that this small RNA molecule has a major effect on phylogeny. The importance of miRNAs is also suggested from recent research demon- strating that miRNA-guided gene regulation is involved in diverse biological functions, such as cell differentiation, development, carcinogenesis, and tumour suppression [3-6]. For example, phylogenetically conserved miRNAs (e. g., let-7, miR-1, miR-124, and miR-125) are involved in cell differentiation and development [7-10]. In this case, let-7 regulates the expression of RAS proteins known as critical oncogene products [11]. Moreover, miR-34, another evolu- tionarily conserved miRNA, is a direct downregulator of © 2010 Takane et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.