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.

项目概要/摘要 在哺乳动物细胞中，mRNA 的转录由 RNA 聚合酶 II (Pol II) 催化，RNA 聚合酶 II 是一种 DNA 聚合酶。 依赖RNA聚合酶。 Pol II 还具有 RNA 依赖性 RNA 聚合酶 (RdRP) 活性；它可以使用RNA 作为模板合成具有确定序列的新RNA。 Pol II RdRP 的功能广泛 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 医学相关；它是导致肝病的丁型肝炎病毒复制所必需的。开创性的 建议进行实验来表征人和小鼠细胞中 Pol II 的 RdRP 活性，并 确定其在控制 RNA 代谢和逆转录转座中的作用。此外，RdRPome – RNA 由 RdRP 活性产生——将在人类和小鼠细胞中被识别。这将是双方首次共同努力 确定 RdRP 活性对哺乳动物细胞中 RNA 群体的影响程度，为 RdRP 生成或修饰的 RNA 的生物学影响和功能的未来研究。 目标 1. 了解 Pol II RdRP 活性如何与 Drosha 和 Dicer 结合来控制 SINE RNA 代谢和正弦逆转录转座。 Pol II RdRP 活性可以延伸 B2 和 Alu RNA 的 3' 末端， 它们是从短散布元件（SINE）转录的。初步数据显示，此次延期 允许 Drosha 甚至 Dicer 切割 RNA。 Pol II RdRP、Drosha 和 Dicer 在 SINE RNA 降解的调节将通过生化和细胞实验来确定。在 此外，将进行研究以了解这三项活动在控制 SINE 中的作用 逆转录转座，需要 RNA 中间体并可能导致疾病。目标 2. 识别人类和 小鼠 RdRPomes 并确定 Pol II RdRP 对选定 RNA 代谢的影响。程度 Pol II 或其他细胞聚合酶作为 RdRP 的功能仍然是一个悬而未决的问题，并且没有系统的研究 已设计出鉴定哺乳动物 RdRP 衍生序列的方法。将开发一种技术 选择性标记细胞中 RdRP 活性产生的 RNA 序列，纯化这些 RNA，并通过 深度测序。由 Pol II 产生的 RdRP 生成的 RNA 将被定义，功能研究将探索 Pol II RdRP 活性是否调节细胞中选定 RNA 的稳定性。 这些研究将测试 Pol II 的作用，这些作用超出了其在传统中心法则中的功能。 RdRP 已被证明在非哺乳动物系统中发挥重要作用；拟议的研究可能揭示关键的 涉及哺乳动物 RdRP 活性的细胞调节机制尚不明确。亲戚 缺乏旨在了解哺乳动物中 RdRP 活性的普遍性和重要性的研究 细胞增强了拟议研究的及时性和意义。