Understanding microRNA Biogenesis and Function

描述(申请人提供)：microRNAs(MiRNAs)是~22nT长的单链RNA引导，调节植物和动物基因的表达。MiRNAs调控着超过三分之一的人类基因，在特定类型的细胞中不适当地产生或丢失microRNAs可能会导致人类疾病。因此，更好地了解miRNAs是如何制造的，以及它们是如何发挥作用的，将促进对人类疾病的病因和潜在治疗方法的了解。这些研究的目的是了解高等真核生物如何利用这些微小的核调节因子及其前体中的信息来确保成熟miRNAs的准确生产，将其装载到功能蛋白质复合体中，以及对真实的mRNA靶标进行适当类型的调控。MiRNAs于1993年被发现，2001年开始对其生物发生的研究，发现多域核糖核酸酶III(RNaseIII)酶将miRNA前体(前miRNAs)转化为成熟的miRNAs。在其间的几年里，已知和预测的miRNAs的数量从两个增加到了10,000多个。作为一类，miRNAs在协调基因表达变化方面的重要性可能与转录因子不相上下。这项建议使用果蝇黑腹果蝇来研究miRNAs的生物发生和组装成功能复合体，以及miRNAs对mRNA靶标的调控，因为这些过程在果蝇和人类之间非常保守。我们将结合果蝇的无细胞生化方法、细胞培养实验、细胞生物学和体内遗传学来剖析miRNA途径的机制和生物学意义。在苍蝇身上学到的东西在哺乳动物细胞提取物中进行测试，并在体外培养的人类和小鼠细胞系中进行测试，以确定这些过程在哪里是保守的，在苍蝇和哺乳动物之间它们的分歧在哪里。下一个资助期将继续努力了解miRNA的生物发生和功能。该项目旨在确定从前miRNA生产miRNA所需的蛋白质和蛋白质复合体，并确定这些蛋白质在miRNA成熟过程中的功能；确定小RNA如何被分类成包含不同ArgAerte蛋白质的复合体；确定如何控制miRNA的稳定性；以及确定跟踪和修剪Ago1结合的miRNAs的酶。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are ~22 nt long, single-stranded RNA guides that regulate the expression of plant and animal genes. miRNAs regulate more than one-third of all human genes, and inappropriate production or loss of microRNAs in specific types of cells can cause human disease. Thus, a better understanding of how miRNAs are made and how they function will advance knowledge of the causes of and potential treatments for human diseases. The goal of these studies is to understand how the information in these tiny riboregulators and their precursors is used by higher eukaryotes to ensure the accurate production of mature miRNAs, their loading into functional protein complexes, and the appropriate type of regulation of authentic mRNA targets. miRNAs were discovered in 1993 and the study of their biogenesis began in 2001 with the discovery that the multi-domain ribonuclease III (RNase III) enzyme Dicer converts miRNA precursors (pre-miRNAs) into mature miRNAs. In the intervening years, the number of known and predicted miRNAs has grown from two to more than 10,000. As a class, miRNAs may rival transcription factors in their importance for orchestrating changes in gene expression. This proposal uses Drosophila melanogaster to study the biogenesis and assembly of miRNAs into functional complexes, as well as the regulation of mRNA targets by miRNAs, because these processes are closely conserved between flies and humans. A combination of cell-free biochemical methods, cell culture experiments, cell biology, and in vivo genetics in flies will be used to dissect the mechanism and biological importance of the miRNA pathway. What is learned in flies is tested in mammalian cell extracts, and in vitro in cultured human and mouse cell lines to identify where these processes are conserved and where they diverge between flies and mammals. The next grant period will continue efforts to understand miRNA biogenesis and function. The project seeks to identify the proteins and protein complexes required to produce miRNA from pre-miRNA and to determine how these proteins function in miRNA maturation; determine how small RNAs are sorted into complexes containing different Argonaute proteins; determine how miRNA stability is controlled; and identify the enzymes that tail and trim Ago1-bound miRNAs.