Functional Roles of Nascent RNA Structure in Regulating and Coordinating Gene Expression

摘要 RNA结构可以影响基因表达的许多方面，包括转录、翻译、剪接、 和多聚腺苷酸化。由于RNA折叠在转录过程中立即发生，这引发了一个基本的 关于新生RNA结构如何影响RNA加工和基因表达的问题。在这里，我们将 通过广泛分布的核糖开关的详细结构-功能研究来解决这个问题的各个方面 与特定配体结合的调控RNA可以控制转录、翻译和剪接。 核糖开关由两个结构域组成-一个高度结构的适体，可以结合特定的配体，以及一个 由于适体配体而改变结构和调节表达的下游表达平台 互动。核糖开关感知一系列代谢物、金属、离子和其他小分子来调节 医学上重要病原体的基本和毒力基因，如单核细胞增多性李斯特菌， 金黄色葡萄球菌和霍乱弧菌，使它们成为新型抗菌药物的靶标 治疗。它们还被开发为用于生物医学应用的新型生物传感器。核糖开关是 强大的模型系统，用于理解RNA生物学的各个领域，包括RNA-配体相互作用， 基因调控机制、细胞核糖核酸结构和基于结构的药物发现。此外，a 许多核糖开关的关键特征是，调节只发生在转录过程中，这使它们成为理想的 研究新生核糖核酸结构对基因表达影响的模型系统。 我们的长期目标是发展对RNA如何共转录的分子理解 折叠调节和协调基因表达和RNA加工，我们正在使用不同的核糖开关 将转录作为模型系统进行调控。该提案详细说明了一系列互补的具体目标， 解决基本问题：(1)什么是序列决定因素和转录动力学 通过共转录链置换促进高效表达平台折叠，以及(2)什么是 适配子-配体相互作用阻止共转录链位移以制定 监管决定。为了解决这些问题，我们将采用功能优先的研究策略，该策略使用 包括FACS-SEQ在内的快速鉴定核糖开关序列的互补方法 细胞中的变体，共转录的SHAPE-SEQ(用引物延伸分析选择性2‘-羟基酰化 测序)以表征核苷酸分辨的配体依赖的共转录折叠，RNA 聚合酶突变体以揭示转录动力学与核糖开关折叠和功能的耦合，以及 研究核糖开关结构-功能联结的计算数据分析方法。详细 共转录RNA折叠如何与新生RNA-配体相互作用和基因调控相联系的知识 将有助于更深入地了解基因表达过程，以及正在进行的努力 开发以RNA为靶点的新疗法，并将RNA工程用于治疗和生物医学应用。

SUMMARY RNA structures can influence many aspects of gene expression including transcription, translation, splicing, and polyadenylation. As RNA folding occurs immediately during transcription, this raises a fundamental question as to how nascent RNA structures influence RNA processing and gene expression. Here we will address aspects of this question through detailed structure-function studies of riboswitches, broadly distributed regulatory RNAs that in response to binding specific ligands can control transcription, translation and splicing. Riboswitches consist of two domains – a highly structured aptamer that can bind a specific ligand, and a downstream expression platform that changes structure and regulates expression due to aptamer-ligand interactions. Riboswitches sense an array of metabolites, metals, ions and other small molecules to regulate essential and virulence genes in medically important pathogens such as Listeria monocytogenes, Staphylococcus aureus and Vibrio cholerae, making them of great interest as targets for novel antimicrobial therapies. They are also being developed as novel biosensors for biomedical applications. Riboswitches are powerful model systems for understanding diverse areas of RNA biology including RNA-ligand interactions, mechanisms of gene regulation, cellular RNA structures, and structure-based drug discovery. In addition, a critical feature of many riboswitches is that regulation only occurs during transcription, making them ideal model systems to study the impacts of nascent RNA structure on gene expression. Towards our long-term goal of developing a molecular understanding of how cotranscriptional RNA folding regulates and coordinates gene expression and RNA processing, we are using diverse riboswitches that regulate transcription as model systems. This proposal details a set of complementary specific aims that address fundamental questions: (1) what are the sequence determinants and transcription dynamics that promote efficient expression platform folding through cotranscriptional strand displacement, and (2) what are the mechanisms by which aptamer-ligand interactions block cotranscriptional strand displacement to enact the regulatory decision. To address these questions, we will apply a ‘function-first’ research strategy that uses complementary approaches including FACS-seq to rapidly functionally characterize riboswitch sequence variants in cells, cotranscriptional SHAPE-Seq (selective 2’-hydroxyl acylation analyzed by primer extension sequencing) to characterize ligand-dependent cotranscriptional folding at nucleotide resolution, RNA polymerase mutants to uncover the coupling of transcription dynamics to riboswitch folding and function, and computational data analysis approaches to study the structure-function linkage in riboswitches. Detailed knowledge of how cotranscriptional RNA folding links to nascent RNA-ligand interactions and gene regulation will contribute to a deeper understanding of gene expression processes, as well as to ongoing efforts to develop new therapeutics that target RNAs and to engineer RNA for therapeutic and biomedical applications.