发展单碱基分辨率的新方法系统鉴定miRNA靶标及其结合位置

About 90% expressed genes in mammalian cells are regulated by miRNA, miRNA recognize its targets through seed region base pairing with 3' untranslated regions of mRNA, and further inhibit mRNA translation or promote mRNA decay. The diversity and evolutionary conservation of miRNA strengthening its functional importance, however, the function of most miRNA have not been determined, especially for those expressed at lower or moderate levels. Currently, the methods applied to identify miRNA targets are achieved by bioinformatics software prediction, but the false positive rate is too high to verify the real targets globally, moreover, there are many conflicts between the functional mechanisms of miRNA revealed at different context. Currently, the main bottleneck in the field is the lack of an unbiased and high resolution experimental methods for the identification of miRNA targets in vivo. This proposal intends to develop a miRACE (miRNA-primed Rapid Amplification of cDNA Ends) method to systematically identify miRNA targets and its in vivo binding sites at physiological conditions, and investigate the different functions of Ago proteins. Identify miRNA target and its precise positions finally may reveal new rules of miRNA in gene regulation, and it also provides a new experimental method for studying the functional mechanism of miRNA in nucleus, the method may also provide a new tools to study other types of non coding RNA.

哺乳动物细胞中约90%的基因受miRNA调节，miRNA通过种子区的6-8个碱基与mRNA的3’UTR配对而识别靶基因, 进而抑制翻译或促进mRNA降解。MiRNA的多样性及进化保守性决定了其功能的重要性，然而大部分低中等水平表达的miRNA功能未知。目前鉴定miRNA靶基因主要是利用生物信息学软件预测，但这种方法假阳性太高、难以大规模实验验证，而且通过生物信息学分析得到的miRNA作用机制与规律多有矛盾。目前困扰miRNA领域的主要问题是缺乏高效的、高分辨率的鉴定miRNA靶标的实验方法。本项目拟开发miRACE方法在细胞水平鉴定所有miRNA的靶基因及其具体结合位置，研究Argonaute家族蛋白在体内的不同功能。精准鉴定miRNA靶基因及其结合位置有望重新揭示miRNA调控基因表达的新规律，同时为研究miRNA在细胞核内的功能机制及其它类型非编码RNA提供新的实验手段。

本项目拟开发miRACE方法在细胞水平鉴定所有miRNA的靶基因及其具体结合位置，研究Argonaute家族蛋白在体内的不同功能。精准鉴定miRNA靶基因及其结合位置有望重新揭示miRNA调控基因表达的新规律，同时为研究miRNA在细胞核内的功能机制及其它类型非编码RNA提供新的实验手段。本项目通过构建稳转细胞系，利用miRACE方法系统鉴定了Ago2在Hela细胞内的所有RNA靶标。通过对比我们的数据和别人发表的数据，进一步的验证了我们方法的可靠性和稳定性，这为本领域的发展作出了重要的贡献。