Beyond the Central Dogma: Investigating Mammalian RNA-dependent RNA Polymerase Activity

 描述（由申请人提供）：RNA聚合酶II（Pol II）被经典地描述为转录真核细胞内所有mRNA的DNA依赖性RNA聚合酶（DdRP）。奇怪的是，Pol II还具有作为RNA依赖性RNA聚合酶（RdRP）的第二功能，使用小鼠B2非编码RNA作为模板和底物，用于向B2 RNA 3'端添加独特的18-nt延伸。这种延伸导致B2 RNA的稳定性和功能改变，表明Pol II RdRP活性可以在转录后水平调节RNA代谢。虽然Pol II DdRP转录的机制和调控已被广泛研究，但对Pol II RdRP 3'延伸的潜在机制步骤、细胞内RdRP活性的大小或RdRP活性对RdRP衍生RNA稳定性的影响知之甚少。本文提出的研究将确定体外和细胞内Pol II RdRP反应所需的B2 RNA二级结构。此外，将开发一种新的RdRP测序（RdRP-seq）方法，该方法将允许检测所有细胞RdRP衍生的RNA。在发现哺乳动物RdRPome的成员后，将鉴定Pol II特异性RdRP产生的RNA，并研究RdRP活性对RNA半衰期的影响。这些研究将为这种独特的Pol II RdRP活性的机制和生物学意义提供有价值的见解。

 DESCRIPTION (provided by applicant): RNA polymerase II (Pol II) is classically described as a DNA-dependent RNA polymerase (DdRP) that transcribes all mRNAs within the eukaryotic cell. Curiously, Pol II also possesses a secondary function as an RNA-dependent RNA polymerase (RdRP), using the mouse B2 non-coding RNA as both a template and a substrate for the addition of a distinct 18-nt extension to the B2 RNA 3' end. This extension results in altered stability and function of B2 RNA, suggesting Pol II RdRP activity may modulate RNA metabolism at the post- transcriptional level. While the mechanism and regulation of Pol II DdRP transcription has been extensively studied, little is known about the underlying mechanistic steps of Pol II RdRP 3' extension, the magnitude of RdRP activity within the cell, or the effects of RdRP activity on the stability of RdRP-derived RNAs. The studies proposed here will identify B2 RNA secondary structures that are required for the Pol II RdRP reaction in vitro and within cells. Furthermore, a novel RdRP-sequencing (RdRP-seq) approach will be developed that will allow detection of all cellular RdRP-derived RNAs. After uncovering members of the mammalian RdRPome, the Pol II-specific RdRP-generated RNAs will be identified, and the effects of RdRP activity on RNA half-life will be investigated. These studies will provide valuable insight into the mechanism and biological significance of this unique Pol II RdRP activity.