Understanding microRNA Biogenesis and Function

描述（由申请人提供）：MicroRNA（miRNA）是约22 nt长的单链RNA向导，可调节植物和动物基因的表达。miRNAs调节超过三分之一的人类基因，在特定类型的细胞中，microRNAs的不适当产生或丢失可能导致人类疾病。因此，更好地了解miRNAs是如何产生的以及它们是如何发挥作用的，将有助于我们进一步了解人类疾病的病因和潜在的治疗方法。这些研究的目的是了解高等真核生物如何使用这些微小的核糖核酸调节子及其前体中的信息来确保成熟miRNA的准确产生，将其加载到功能蛋白质复合物中，以及对真实mRNA靶点进行适当类型的调节。miRNA于1993年被发现，其生物起源的研究始于2001年，发现多结构域核糖核酸酶III（RNase III）酶Dicer将miRNA前体（pre-miRNA）转化为成熟的miRNA。在此期间的几年里，已知和预测的miRNA数量已从两种增加到超过10，000种。作为一类，miRNAs在协调基因表达变化方面的重要性可与转录因子相媲美。该提案使用果蝇来研究miRNAs的生物发生和组装成功能复合物，以及miRNAs对mRNA靶标的调节，因为这些过程在果蝇和人类之间非常保守。结合无细胞生物化学方法，细胞培养实验，细胞生物学，和在苍蝇体内遗传学将被用来解剖的机制和生物学的重要性的miRNA途径。在果蝇中学到的东西在哺乳动物细胞提取物中进行了测试，并在体外培养的人类和小鼠细胞系中进行了测试，以确定这些过程在果蝇和哺乳动物之间的保守性和差异。下一个资助期将继续努力了解miRNA的生物起源和功能。该项目旨在确定从pre-miRNA产生miRNA所需的蛋白质和蛋白质复合物，并确定这些蛋白质在miRNA成熟中的功能;确定小RNA如何被分选成含有不同Argonaute蛋白的复合物;确定如何控制miRNA稳定性;并确定尾和修剪Ago 1结合的miRNA的酶。 公共卫生相关性： MicroRNA调节着超过三分之一的人类基因，在特定类型的细胞中，这些微小RNA的不适当产生或丢失可能导致人类疾病。果蝇的microRNA通路与哺乳动物的microRNA通路高度相似，因此这些研究中所学到的将促进我们对人类microRNA的理解。因此，拟议的研究有望有助于更好地理解和治疗人类疾病。

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. PUBLIC HEALTH RELEVANCE: MicroRNAs regulate more than one-third of all human genes, and inappropriate production or loss of these tiny RNAs in specific types of cells can cause human disease. The Drosophila microRNA pathway is highly similar to its mammalian counterpart, so what is learned in these studies will advance our understanding of microRNAs in humans. Thus, the proposed studies promise to contribute to a better understanding of and therapy for human diseases.