Understanding microRNA Biogenesis and Function

了解 microRNA 的生物发生和功能

• 批准号: 8041203
• 负责人: PHILLIP D ZAMORE
• 金额: $ 51.91万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8041203
• 关键词: Animals; Binding; Biochemical; Biogenesis; Biological; Cell Culture Techniques; Cell Extracts; Cells; Cellular biology; Complex; Cultured Cells; Data; Dicer Enzyme; Drosophila genus; Drosophila melanogaster; Ensure; Enzymes; Eukaryota; Gene Expression; Genes; Genetic; Genetic Transcription; Goals; Grant; Guide RNA; Human; In Vitro; Knowledge; Learning; Mammalian Cell; Mammals; Messenger RNA; Methods; MicroRNAs; Modification; Molecular; Mouse Cell Line; Nucleotides; Pathway interactions; Plants; Process; Production; Protein Isoforms; Proteins; Publishing; Regulation; Ribonuclease III; Small RNA; Sorting - Cell Movement; Structure; Tail; Testing; Transferase; cell type; endonuclease; fly; human DICER1 protein; human disease; in vivo; mRNA Expression; nuclease; pre-miRNA; protein complex; protein function; research study; response; tool; transcription factor

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.

描述（由申请人提供）：MicroRNAs （miRNAs）是一种长约22 nt的单链RNA向导，可调节植物和动物基因的表达。microrna调节着超过三分之一的人类基因，在特定类型的细胞中，microrna的不适当产生或缺失会导致人类疾病。因此，更好地了解mirna是如何产生的以及它们是如何起作用的，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这些微小的核糖体调节剂及其前体中的信息如何被高等真核生物利用，以确保成熟mirna的准确产生，将其装载到功能性蛋白质复合物中，以及对真实mRNA靶标的适当类型的调节。miRNAs于1993年被发现，2001年发现多结构域核糖核酸酶III （RNase III）酶Dicer将miRNA前体（pre-miRNAs）转化为成熟miRNAs，开始了对其生物发生的研究。在这中间的几年里，已知和预测的mirna数量已经从2个增加到1万多个。作为一类，mirna在调控基因表达变化方面的重要性可能与转录因子相媲美。由于这些过程在果蝇和人类之间是密切保守的，因此本提案利用果蝇研究mirna的生物发生和组装成功能复合物，以及mirna对mRNA靶点的调控。将结合无细胞生化方法、细胞培养实验、细胞生物学和果蝇体内遗传学来剖析miRNA通路的机制和生物学重要性。在苍蝇身上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，以确定这些过程在苍蝇和哺乳动物之间哪些地方是保守的，哪些地方是不同的。下一个资助期将继续努力了解miRNA的生物发生和功能。该项目旨在鉴定从pre-miRNA产生miRNA所需的蛋白质和蛋白质复合物，并确定这些蛋白质如何在miRNA成熟过程中发挥作用；确定小rna如何被分类成含有不同Argonaute蛋白的复合物；确定如何控制miRNA的稳定性；并鉴定出与ago1结合的mirna尾部和修剪的酶。