Computational prediction and experimental validation of evolutionarily conserved microRNA target genes in bilaterian animals.
Computational prediction and experimental validation of evolutionarily conserved microRNA target genes in bilaterian animals.
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DOI：
10.1186/1471-2164-11-101
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发表时间：
2010-02-09
期刊：
影响因子：
4.4
通讯作者：
Kanai A
中科院分区：
文献类型：
--
作者：
Takane K;Fujishima K;Watanabe Y;Sato A;Saito N;Tomita M;Kanai A
在许多真核生物中，微小RNA（miRNAs）与靶信使RNA（mRNAs）的3 '-非翻译区（3'-UTR）中的互补位点结合，并在翻译阶段调节其表达。最近的研究表明，许多miRNAs在进化上是保守的;然而，其靶基因的进化尚未得到系统的表征。我们试图阐明一组保守的miRNA/靶基因对，并分析双边进化早期阶段miRNA介导的基因调控机制。最初，我们在五种不同的双侧性动物中提取了五种进化上保守的miRNA（let-7、miR-1、miR-124、miR-125/lin-4和miR-34）。随后，我们设计了一个程序来预测进化保守的miRNA/靶基因对，通过引入直向同源基因信息。结果，我们提取了31个在至少四种不同的双侧性动物中保守的正向miRNA/靶基因对;预测集显示出实验验证的正向miRNA/靶基因对的显著富集。大约84%的靶基因由三种miRNA（let-7、miR-1和miR-124）调控，它们的功能主要分为以下几类：发育、肌肉形成、细胞粘附和基因调控。我们使用报告基因测定来实验验证HeLa细胞中六个候选对（六个测试对）的下调。我们的新方法的应用使31个miRNA/靶基因对的鉴定成为可能，这些miRNA/靶基因对被认为是从共同的双侧祖先时代开始受到调控的。所有六个候选对的下调表明，正向同源信息有助于阐明由miRNA调控的原始基因集;它也是消除预测候选人假阳性的有效工具。总之，我们的研究确定了潜在的重要的miRNA-靶对，这些对在不同的两侧对称动物中进化上保守，并可能为早期miRNA功能提供新的见解。
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. 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. 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.
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