Function and evolution of microRNAs

描述(申请人提供)：最近的实验表明，蠕虫、苍蝇、人类和老鼠等生物的基因组编码数百个新的microRNA基因。许多这些小的非编码基因被认为通过与目标信使RNA中的部分互补位点结合来调节其他基因的翻译表达。最初的表型和表达分析表明，microRNAs在发育、病毒应答和凋亡等基本细胞过程中发挥着重要作用。此外，许多microRNA在很大的进化距离内是保守的。然而，大多数microRNAs的生物学功能尚不清楚。尽管最近的计算方法在识别microRNA靶标方面取得了进展，但还没有实验上的高通量方法来剖析microRNA功能。我们的长期目标是建立一个生物信息学和实验方法相结合的系统，使高通量组合分析microRNA功能成为可能。在这里，我们建议设置这个系统。具体地说，我们将(A)在硅胶中使用方法来预测五种不同线虫物种中的microRNA靶标，(B)使用抑制寡核苷酸在体内敲除几种线虫物种中20个microRNA的功能，并登记它们在整个发育过程中的功能丧失表型，(C)开发一个系统来验证体内预测的microRNA靶标，并测试25个计算预测的靶标。这些数据将对microRNAs的生物功能及其在塑造生物多样性方面的潜在作用产生新的见解。这个项目是生物信息学实验室(专注于基因调控过程)和实验实验室(线虫的RNAi功能基因组学)之间的密集合作。

DESCRIPTION (provided by applicant): Recent experiments have shown that the genomes of organisms such as worm, fly, human, and mouse encode hundreds of novel microRNA genes. Many of these small non-coding genes are thought to regulate the translational expression of other genes by binding to partially complementary sites in target messenger RNAs. Initial phenotypic and expression analyses suggest an important role for microRNAs in basic cellular processes such as development, viral response, and apoptosis. Furthermore, many microRNAs are conserved over large evolutionary distances. However, the biological function of most microRNAs is unknown. Although recent computational methods have made progress towards the identification of microRNA targets, no experimental high-throughput method for dissecting microRNA function exists. Our long-term goal is to create a system where bioinformatic and experimental methods are integrated to make possible the high-throughput combinatorial analysis of microRNA function. Here, we propose to setup this system. Specifically, we will (a) employ in silica methods to predict microRNA targets in five different nematode species, (b) use inhibitor oligonucleotides to knock down the function of 20 microRNAs across several nematode species in vivo, and register their loss-of-function phenotypes throughout development, (c) develop a system to validate predicted microRNA targets in vivo and test 25 computationally predicted targets. These data will generate novel insights into the biological function of microRNAs and their potential role in shaping biological diversity. This project is an intensive collaboration between a bioinformatics lab (focused on gene regulatory processes), and an experimental lab (RNAi functional genomics in nematodes)