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（由适用提供）：在生物学中出现了一种新的范式，其中RNA分子是积极参与者在确定，催化和控制基本细胞过程中 - 直到最近为蛋白质保留的角色。两个新兴主题特别有趣，并且一直是我实验室的研究重点。第一个主题涉及RNA作为指导，即信息载体，以指导蛋白质对核酸靶标的作用。 RNAi和CRISPR-CA的体现的这种系统的功率可以用于治疗以及基因组工程应用。到目前为止，已经在88％的古细胞基因组和39％的细菌基因组中鉴定了CRISPR-CAS防御系统，包括重要的人类病原体，例如弯曲杆菌的人类空肠，肉毒杆菌，埃希氏梭菌，大肠杆菌，单细胞增生核细胞儿童，乳腺细胞增生，乳腺菌菌，结膜菌和yersiaia pestis。已显示它可以调节水平基因转移和生物膜形成。我们提出的项目1基于几种重要的CAS蛋白的成功结构确定以及从B. halodurans的I型Cascade复合物的成功重建。在此提案中，我们提出了实验，以了解I型I-C CRISPR- CAS系统中的CRIS PRTRENEFERCE机制。我们以强大的初步数据为基础（1）表征了I型I-C系统中目标搜索级联复合物的结构功能，（2）表征了级联相互作用蛋白Cas3的结构功能，这是所有I型I型CRISPR-CAS系统中的重要因素。 （3）捕获喀斯喀特-DSDNA和CASCADE-CAS3复合物的结构快照。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械多样性。 RNA生物学中的第二个中心主题涉及在顺式中执行基因调节功能的结构化RNA。所谓的核糖开关的短顺式作用RNA元件的发现导致基因调节概念的范式转移。核糖开关在原核生物中是宽度的，据估计它们可以控制富富基因中所有基因的2-4％。它们几乎仅在顺式中起作用，通常驻留在宿主mRNA的5'未翻译区域（5'- UTR）中，并主要通过过早转录终止或抑制翻译起始的方式来调节基因表达，尽管其他调节机制在内 已经证明了mRNA裂解，稳定性和替代剪接。我们确定了核糖开关研究中的以下前沿，并相应地使我们的努力保持一致：1。新颖的配体灵敏度策略利用了核糖开关，该研究可能揭示了细菌生理学的新方面（项目2中T盒核糖开关的研究）； 2。对构象开关机制的更深入了解（项目3中的YYBP-YKOY孤儿核糖开关）； 3。孤儿核糖开关家庭的结构功能表征（项目3）；和4。在工业，医学，药房或环境保护中的合成生物学应用（项目3中的荧光MN2+传感器应用）。