Mechanistic investigation of RNA-mediated gene regulation and immunity

RNA介导的基因调控和免疫的机制研究

• 批准号: 9976558
• 负责人: Ailong Ke
• 金额: $ 59.86万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9976558
• 关键词: 5' Untranslated Regions; Alternative Splicing; Antibiotics; Archaeal Genome; BCAR1 gene; Bacteria; Bacterial Genome; Bacterial Physiology; Biology; CRISPR interference; Campylobacter; Cell physiology; Clostridium botulinum; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Data; Development; Elements; Environmental Protection; Escherichia coli; Essential Genes; Family; Firmicutes; Gene Expression; Gene Expression Regulation; Genes; Genome engineering; Horizontal Gene Transfer; Human; Immunity; Industry; Investigation; Ligands; Listeria monocytogenes; Mediating; Medicine; Messenger RNA; Microbial Biofilms; Molecular Conformation; Mycobacterium tuberculosis; Nucleic Acids; Operon; Orphan; Participant; Pharmacy facility; Prokaryotic Cells; Proteins; RNA; RNA Interference; Regulator Genes; Research; Role; Structure; System; Therapeutic; Translation Initiation; Untranslated Regions; Virulence; Yersinia pestis; base; ds-DNA; experimental study; fascinate; frontier; genome editing; human pathogen; novel; pathogenic microbe; premature; public health relevance; reconstitution; sensor; synthetic biology; transcription termination

 DESCRIPTION (provided by applicant): A new paradigm has emerged in biology in which RNA molecules are active participants in regulating, catalyzing and controlling fundamental cellular processes - roles that were reserved for proteins until recently. Two emerging themes are particularly fascinating and have been the research focus in my lab. The first theme involves RNA serving as a guide, an information carrier, to direct the action of proteins on nucleic acid targets. The power in such systems, exemplified by RNAi and CRISPR-Cas, can be harnessed for therapeutics as well as genome engineering applications. CRISPR-Cas defense systems have been identified in 88% of archaeal genomes and 39% of bacterial genomes thus far sequenced, including important human pathogens such as Campylobacter human jejuni, Clostridium botulinum, Escherichia coli, Listeria monocytogenes, Mycobacterium tuberculosis and Yersinia pestis. It has been shown to modulate the horizontal gene transfer and biofilm formation. Our proposed Project 1 is based on the successful structure determination of several important Cas proteins and the successful reconstitution of the Type I-C Cascade complex from B. halodurans. In this proposal, we propose experiments to understand the CRISPR interference mechanism in Type I-C CRISPR- Cas system. We build upon strong preliminary data to (1) characterize the structure-function of the target searching Cascade complex in Type I-C system, (2) characterize the structure-function of the Cascade- interacting protein Cas3, an essential factor in all Type I CRISPR-Cas systems. (3) capture structure snapshots of the Cascade-dsDNA and the Cascade-Cas3 complexes. Our finding will serve to reveal the common theme and mechanistic diversity among different CRISPR-Cas systems. The second central theme in RNA biology involves structured RNAs performing gene regulatory function in cis. The discovery of short cis- acting RNA elements termed riboswitches led to a paradigm shift in the concept of gene regulation. Riboswitches are widespread in prokaryotes, where they are estimated to control as many as 2-4% of all genes in Firmicutes. They almost exclusively function in cis, usually reside in the 5' untranslated regions (5'- UTRs) of the host mRNAs, and regulate gene expression mainly through the means of premature transcription termination or inhibition of translation initiation, although other regulatory mechanisms including the control of mRNA cleavage, stability, and alternative splicing have been demonstrated. We identify the following frontiers in the riboswitch research and align our efforts accordingly: 1. novel ligand sensing strategy utilized riboswitches, the study of which may reveal novel aspects of bacterial physiology (the study of T box riboswitches in Project 2); 2. deeper understanding of the conformational switching mechanism (the yybP-ykoY orphan riboswitches in Project 3); 3. structure-function characterization of orphan riboswitch families (Project 3); and 4. synthetic biology applications in industry, medicine, pharmacy or environmental protection (fluorescent Mn2+ sensor applications in Project 3).

 描述（由申请人提供）：生物学中出现了一种新的范例，其中RNA分子是调节、催化和控制基本细胞过程的积极参与者-直到最近才保留给蛋白质的角色。两个新兴的主题特别吸引人，一直是我实验室的研究重点。第一个主题涉及RNA作为向导，信息载体，指导蛋白质对核酸靶点的作用。以RNAi和CRISPR-Cas为例，这种系统的力量可以用于治疗和基因组工程应用。CRISPR-Cas防御系统已经在88%的古细菌基因组和39%的细菌基因组中被鉴定，包括重要的人类病原体，如空肠弯曲杆菌、肉毒梭菌、大肠杆菌、单核细胞增生李斯特菌、结核分枝杆菌和鼠疫耶尔森菌。已显示其调节水平基因转移和生物膜形成。我们提出的项目1是基于几种重要Cas蛋白的成功结构测定和来自B的I-C型级联复合物的成功重建。耐盐生物。在该提案中，我们提出了实验来理解I-C型CRISPR-Cas系统中的CRISPR干扰机制。我们基于强有力的初步数据来（1）表征I-C型系统中靶搜索Cascade复合物的结构-功能，（2）表征Cascade相互作用蛋白Cas 3的结构-功能，Cas 3是所有I型CRISPR-Cas系统中的必需因子。(3)Cascade-dsDNA和Cascade-Cas 3复合物的捕获结构快照。我们的发现将有助于揭示不同CRISPR-Cas系统之间的共同主题和机制多样性。RNA生物学的第二个中心主题涉及结构化RNA顺式执行基因调控功能。被称为核糖开关的短顺式作用RNA元件的发现导致了基因调控概念的范式转变。核糖开关广泛存在于原核生物中，据估计它们控制着厚壁菌门中多达2-4%的基因。它们几乎完全以顺式发挥功能，通常位于宿主mRNA的5'非翻译区（5'-UTR），并且主要通过过早转录终止或抑制翻译起始的方式调节基因表达，尽管其他调节机制包括控制转录终止或抑制翻译起始。 mRNA的切割、稳定性和可变剪接已得到证实。我们确定了核糖开关研究的以下前沿领域，并相应地调整我们的努力：1。新的配体传感策略利用核糖开关，其研究可以揭示细菌生理学的新方面（项目2中的T盒核糖开关的研究）; 2.更深入地理解构象转换机制（项目3中的yybP-ykoY孤儿核糖开关）; 3.孤儿核糖开关家族的结构-功能表征（项目3）;和4.合成生物学在工业、医学、制药或环境保护中的应用（项目3中的荧光Mn 2+传感器应用）。