Dissecting the regulatory role of a eukaryotic transcription factor in RNA-templated transcription catalyzed by DNA-directed RNA polymerase II

剖析真核转录因子在 DNA 指导的 RNA 聚合酶 II 催化的 RNA 模板转录中的调节作用

基本信息

批准号：
10047065
负责人：
Ying Wang
金额：
$ 41.09万
依托单位：
MISSISSIPPI STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2023-07-31
项目状态：
已结题

项目摘要

Project Summary Transcription is a fundamental process that mediates the interplay between genetic information and phenotypes, thus vital for development, responses to environmental cues, and diseases. Transcription is primarily catalyzed by DNA-directed RNA polymerases (DdRPs), which are highly conserved among eukaryotic organisms. It has been well established that regulation from multiple layers controls DdRP- catalyzed transcription on DNA templates. Interestingly, some DdRPs recognize both DNA and RNA templates for transcription. This RNA-templated activity regulates gene expression in bacteria and mammalian cells and is employed by pathogens (i.e., viroids and human hepatitis delta virus) for propagation. However, the machinery and mechanism for RNA-templated transcription by DdRPs are poorly understood. Using the replication of potato spindle tuber viroid (PSTVd) as a model, preliminary data showed that a highly conserved glycine in the second largest subunit of Pol II is critical for DNA-directed but not RNA-templated transcription, indicating the different requirements of this residue for DNA and RNA templates. In addition, data showed that TFIIS, a general transcription factor essential for DNA-directed transcription, is dispensable for PSTVd RNA-templated transcription, suggesting that a specialized group of factors are required for transcription of DNA versus RNA templates. Furthermore, the eukaryotic transcription factor TFIIIA-7ZF, which binds RNA but not DNA, has been shown as a critical factor in facilitating Pol II-catalyzed transcription on PSTVd RNA templates. The TFIIIA-7ZF binding site maps to a region next to the transcription initiation site and is critical for Pol II binding and PSTVd replication, suggesting that this binding site acts as an RNA promoter. Together, these findings inspire the central hypothesis that Pol II recruits a specialized group of factors to selectively recognize RNA as templates and catalyze transcription. Using a novel in vitro transcription (IVT) assay that is based on Pol II-catalyzed transcription of PSTVd, the goal of this project is to further dissect the regulatory mechanism for specialized Pol II machinery to transcribe RNA templates. To achieve this goal, three aims are proposed to further dissect the role of TFIIIA-7ZF in RNA-templated transcription, map the TFIIIA-7ZF/Pol II binding domains, and characterize the molecular basis of an RNA promoter. The expected outcomes will provide novel insights into the regulatory mechanism underlying RNA- templated transcription catalyzed by Pol II and provide valuable knowledge to better delineate RNA promoters. As this project aims to uncover the basic principles governing the modus operandi of RNA polymerases that are highly conserved across eukaryotic organisms, the results gained here will provide an in-depth knowledge of RNA-templated transcription. Such in-depth knowledge will lead to the full understanding of factors and the regulatory mechanism underlying RNA-templated transcription and facilitate the development of effective treatments for diseases caused by infectious RNAs.

项目概要转录是介导遗传信息与遗传信息之间相互作用的基本过程。表型，因此对于发育、对环境线索的反应和疾病至关重要。转录是主要由 DNA 指导的 RNA 聚合酶 (DdRP) 催化，DdRP 在生物体中高度保守真核生物。众所周知，多层监管控制着 DdRP- DNA 模板上的催化转录。有趣的是，一些 DdRP 可识别 DNA 和 RNA 转录模板。这种以 RNA 为模板的活性调节细菌中的基因表达哺乳动物细胞，并被病原体（即类病毒和人类丁型肝炎病毒）用来繁殖。然而，人们对 DdRP 以 RNA 为模板的转录的机制和机制知之甚少。以马铃薯纺锤块茎类病毒（PSTVd）的复制为模型，初步数据表明，高度 Pol II 第二大亚基中的保守甘氨酸对于 DNA 导向而非 RNA 模板至关重要转录，表明该残基对DNA和RNA模板的不同要求。此外，数据表明，TFIIS作为DNA定向转录所必需的通用转录因子，是可有可无的 PSTVd RNA 模板转录，表明需要一组专门的因子 DNA 与 RNA 模板的转录。此外，真核转录因子 TFIIIA-7ZF，它结合 RNA 但不结合 DNA，已被证明是促进 Pol II 催化转录的关键因素在 PSTVd RNA 模板上。 TFIIIA-7ZF 结合位点映射到转录起始附近的区域位点，对于 Pol II 结合和 PSTVd 复制至关重要，表明该结合位点充当 RNA 发起人。总之，这些发现激发了一个中心假设：Pol II 招募了一个专门的团队选择性识别 RNA 作为模板并催化转录的因子。使用体外小说基于 Pol II 催化 PSTVd 转录的转录 (IVT) 测定，该项目的目标是进一步剖析专门的 Pol II 机器转录 RNA 模板的调控机制。到为了实现这一目标，提出了三个目标来进一步剖析 TFIIIA-7ZF 在 RNA 模板化中的作用转录、绘制 TFIIIA-7ZF/Pol II 结合域图并表征 RNA 的分子基础发起人。预期结果将为 RNA 调控机制提供新的见解 Pol II 催化的模板化转录为更好地描绘 RNA 启动子提供了宝贵的知识。由于该项目旨在揭示控制 RNA 聚合酶操作方式的基本原理，在真核生物中高度保守，这里获得的结果将提供深入的知识 RNA 模板转录。这种深入的知识将导致对因素和问题的充分理解。 RNA模板转录的调控机制并促进有效的开发治疗由传染性 RNA 引起的疾病。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Current view and perspectives in viroid replication.

类病毒复制的当前观点和观点。

DOI：
发表时间：
2021
期刊：
影响因子：
0
作者：
Wang; Ying
通讯作者：
Ying

Emerging value of the viroid model in molecular biology and beyond.

类病毒模型在分子生物学及其他领域的新兴价值。

DOI：
10.1016/j.virusres.2022.198730
发表时间：
2022-05
期刊：
Virus research
影响因子：
5
作者：
Ma J;Mudiyanselage SDD;Wang Y
通讯作者：
Wang Y

A nuclear import pathway exploited by pathogenic noncoding RNAs.

致病性非编码 RNA 利用的核输入途径。

DOI：
发表时间：
2022-09-27
期刊：
影响因子：
0
作者：
Ma, Junfei;Dissanayaka Mudiyanselage, Shachinthaka D;Park, Woong June;Wang, Mo;Takeda, Ryuta;Liu, Bin;Wang, Ying
通讯作者：
Wang, Ying

Studies on Viroid Shed Light on the Role of RNA Three-Dimensional Structural Motifs in RNA Trafficking in Plants.

类病毒的研究揭示了 RNA 三维结构基序在植物 RNA 贩运中的作用。