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

项目总结/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。 依赖的RNA聚合酶。Pol II还具有RNA依赖性RNA聚合酶（RdRP）活性;它可以利用RNA 作为模板来合成新的RNA。Pol II RdRP的功能范围 在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;它是导致肝脏疾病的丁型肝炎病毒复制所必需的。开创性 提出了实验来表征Pol II在人和小鼠细胞中的RdRP活性， 确定其在控制RNA代谢和反转录转座中的作用。此外，RdRPome -RNA 由RdRP活性产生-将在人类和小鼠细胞中鉴定艾德。这将是第一次共同努力， 确定RdRP活性在多大程度上影响哺乳动物细胞中的RNA群体，为以下研究铺平道路： RdRP产生或修饰的艾德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结合以控制SINE RNA 代谢和SINE反转录转座。Pol II RdRP活性可以延伸B2和Alu RNA的3'末端， 其转录自短散布元件（西内斯）。初步数据显示， 允许RNA被Drosha或者Dicer切割。Pol II RdRP、Drosha和Dicer在 将使用生物化学和基于细胞的实验来确定调节SINE RNA的降解。在 此外，还将进行研究，以了解这三种活动在控制SINE中的作用 逆转录转座，需要RNA中间体，可导致疾病。目标二。找到所需的人力和 小鼠RdRPomes，并确定Pol II RdRP对所选RNA代谢的影响。程度 Pol II或其他细胞聚合酶作为RdRPs的功能仍然是一个开放的问题，没有系统的 已经设计了鉴定哺乳动物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产生的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择RNA的稳定性。 这些研究将测试Pol II在传统中心法则中超越其功能的作用。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活性的尚未表征的细胞调节机制。的相对 缺乏旨在了解RdRP活性在哺乳动物中的普遍性和重要性的研究 细胞放大了拟议研究的及时性和重要性。

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.