Regulation of RNA Metabolism by the RNA-dependent RNA Polymerase Activity of Mammalian RNA polymerase II

哺乳动物 RNA 聚合酶 II 的 RNA 依赖性 RNA 聚合酶活性对 RNA 代谢的调节

• 批准号: 9335918
• 负责人: James Goodrich
• 金额: $ 33.32万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-19 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9335918
• 关键词: Affect; Biochemical; Biological; Biological Assay; Biology; Cells; Cellular Stress Response; Cleaved cell; Communities; Couples; DNA Polymerase II; DNA Polymerase III; DNA-Directed RNA Polymerase; Data; Dicer Enzyme; Disease; Enzymes; Future; Gene Expression; Genetic Transcription; Hepatitis Delta Virus; Human; Label; Learning; Liver diseases; Mammalian Cell; Medical; Messenger RNA; Metabolism; Methods; MicroRNAs; Modeling; Modification; Mus; Pathway interactions; Play; Polymerase; Population; Prevalence; Process; Publishing; RNA; RNA Polymerase III; RNA Processing; RNA Sequences; RNA Stability; RNA chemical synthesis; RNA-Directed DNA Polymerase; RNA-Directed RNA Polymerase; Regulation; Research; Retrotransposition; Retrotransposon; Role; Seminal; Short Interspersed Nucleotide Elements; Somatic Cell; System; Techniques; Testing; Time; Transcript; Transcription Repressor/Corepressor; Untranslated RNA; Work; base; cell growth regulation; deep sequencing; experimental study; information model; interest; knock-down; mammalian genome; mouse genome; novel; overexpression; reconstitution

PROJECT SUMMARY/ABSTRACT In mammalian cells, transcription of mRNA is catalyzed by RNA polymerase II (Pol II), a DNA- dependent RNA polymerase. Pol II also has RNA-dependent RNA polymerase (RdRP) activity; it can use RNA as a template to synthesize new RNA of defined sequence. The breadth of functions for the Pol II RdRP activity in mammalian cells is unknown, yet could have broad biological significance. Pol II RdRP activity is medically relevant; it is required for replication of hepatitis delta virus, which causes liver disease. Seminal experiments are proposed to characterize the RdRP activity of Pol II in human and mouse cells and to determine its role in controlling RNA metabolism and retrotransposition. In addition, the RdRPome – the RNAs made by RdRP activity – will be identified in human and mouse cells. This will be the first concerted effort to determine the extent to which RdRP activity affects the RNA population in mammalian cells, paving the way for future studies of the biological impacts and functions of RdRP-generated or modified RNAs. Aim 1. Understand how the Pol II RdRP activity couples with Drosha and Dicer to control SINE RNA metabolism and SINE retrotransposition. Pol II RdRP activity can extend the 3' ends of B2 and Alu RNAs, which are transcribed from short interspersed elements (SINEs). Preliminary data suggest that this extension allows the RNAs to be cleaved by Drosha and perhaps Dicer. The roles of the Pol II RdRP, Drosha and Dicer in regulating the degradation of SINE RNAs will be determined using biochemical and cell-based experiments. In addition, studies will be performed to understand the roles of these three activities in controlling SINE retrotransposition, which requires an RNA intermediate and can cause disease. Aim 2. Identify the human and mouse RdRPomes and determine the effect of the Pol II RdRP on the metabolism of select RNAs. The extent to which Pol II or other cellular polymerases function as RdRPs remains an open question, and no systematic method to identify mammalian RdRP-derived sequences has been devised. A technique will be developed that selectively labels RNA sequences made by RdRP activity in cells, purifies these RNAs, and identifies them via deep sequencing. The RdRP-generated RNAs made by Pol II will be defined, and functional studies will probe whether the Pol II RdRP activity regulates the stability of select RNAs in cells. The studies will test roles for Pol II that go beyond its function in the traditional central dogma. RdRPs have been shown to play important roles in non-mammalian systems; the proposed studies could reveal critical yet uncharacterized mechanisms of cellular regulation involving mammalian RdRP activity. The relative absence of studies aimed at understanding the prevalence and importance of RdRP activities in mammalian cells amplifies the timeliness and significance of the proposed research.

项目摘要/摘要 在哺乳动物细胞中，mRNA的转录是由RNA聚合酶II(Pol-II)催化的，它是一种DNA- 依赖的RNA聚合酶。POL-II也具有RNA依赖的RNA聚合酶(RdRP)活性；它可以使用RNA 以此为模板合成DefiNed序列的新核糖核酸。Pol II RdRP的功能广度 哺乳动物细胞中的活性尚不清楚，但可能具有广泛的生物学意义。POL II RdRP活性为 与医学相关；它是复制丁型肝炎病毒所必需的，丁型肝炎病毒会导致肝脏疾病。开创性 本实验旨在研究Pol II在人和小鼠细胞中的RdRP活性，并对其活性进行研究。 确定其在控制RNA新陈代谢和逆转录转座中的作用。此外，RdRPome-RNAs 由Rdrp活性制成-将在人和小鼠细胞中被鉴定为fi。这将是fi第一次共同努力 确定RdRP活性对哺乳动物细胞中RNA群体的影响程度，为 Rdrp产生的或经fi修饰的RNA的生物学影响和功能的未来研究。 目的1.了解POL II RdRP活性如何与DROSHA和DICER偶联来控制Sine RNA 新陈代谢和正弦反转位。Pol II RdRP活性可延长B2和Alu RNA的3‘端， 它们是从短的散布元素(Sine)转录而来的。初步数据显示，这一延期 允许RNAs被DROSHA或DICER切割。POL II RdRP、DROSHA和DICER在蛋白质合成中的作用 调节Sine RNA的降解将通过生化和基于细胞的实验来确定。在……里面 此外，还将进行研究，以了解这三种活动在控制正弦方面的作用 逆转录转座，这需要一种RNA中间体，可以导致疾病。目标2.确认人类和 小鼠RdRPome并确定Pol II RdRP对特定RNA代谢的影响。广度 POLII或其他细胞聚合酶作为RdRPs的作用仍然是一个悬而未决的问题，也没有系统的 已经设计了一种识别哺乳动物Rdrp衍生序列的方法。将开发一种技术， 选择性地标记由细胞中的Rdrp活性产生的rna序列，纯化这些rna，并通过 深度测序。由POL II制造的Rdrp产生的RNA将被defiNed，并将进行功能研究 Pol II RdRP活性是否调节细胞中特定RNA的稳定性。 这些研究将测试波尔II在传统中央教条中超越其功能的角色。RDRPS 已经被证明在非哺乳动物系统中发挥着重要作用；拟议的研究可能揭示出关键的 然而，涉及哺乳动物Rdrp活性的细胞调控机制尚不明确。相对的 缺乏旨在了解RdRP活动在哺乳动物中的盛行率和重要性的研究 Cellsfi增强了这项研究的及时性和重要性。