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

总结 RNA结构可以影响基因表达的许多方面，包括转录、翻译、剪接， 和聚腺苷酸化。由于RNA折叠在转录过程中立即发生，这引起了一个基本的问题。 关于新生RNA结构如何影响RNA加工和基因表达的问题。这里我们将 通过对广泛分布的核糖开关的详细结构-功能研究， 调节RNA响应于结合特异性配体可以控制转录、翻译和剪接。 核糖开关由两个结构域组成，一个是可以结合特定配体的高度结构化的适体， 由于适体-配体而改变结构并调节表达的下游表达平台 交互.核糖开关感知一系列代谢物、金属、离子和其他小分子， 医学上重要的病原体如单核细胞增生李斯特菌中的必需基因和毒力基因， 金黄色葡萄球菌和霍乱弧菌，使他们作为新的抗菌剂的目标非常感兴趣 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生物学的不同领域，包括RNA-配体相互作用， 基因调控机制、细胞RNA结构和基于结构的药物发现。另外还有按 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RNA结构对基因表达的影响。 我们的长期目标是从分子水平上理解共转录RNA是如何 折叠调节和协调基因表达和RNA加工，我们使用不同的核糖开关 作为模型系统来调节转录。该提案详细列出了一套补充性的具体目标， 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有效的表达平台折叠，以及（2） 适体-配体相互作用阻断共转录链置换以实现转录的机制， 监管决定。为了解决这些问题，我们将采用“功能第一”的研究策略， 包括FACS-seq的互补方法来快速功能性地表征核糖开关序列 细胞中的变体，共转录SHAPE-Seq（通过引物延伸分析的选择性2 '-羟基酰化 测序），以表征核苷酸分辨率下的配体依赖性共转录折叠，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偶联，以及 计算数据分析方法来研究核糖开关的结构-功能联系。详细 共转录RNA折叠如何连接到新生RNA-配体相互作用和基因调控的知识 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， 开发靶向RNA的新疗法，并将RNA工程化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.