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

基本信息

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
项目类别：
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&apos 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

项目摘要

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式，其中 RNA 分子积极参与调节、催化和控制基本的细胞过程——直到最近，两个新兴主题特别令人着迷，并且成为了蛋白质的角色。我实验室的研究重点是 RNA 作为信息载体，引导蛋白质对核酸靶点的作用，以 RNAi 和 CRISPR-Cas 为代表的此类系统的力量可用于治疗。迄今为止，已在 88% 的古菌基因组和 39% 的细菌基因组中发现了 CRISPR-Cas 防御系统，其中包括重要的人类病原体，如人类空肠弯曲杆菌、肉毒杆菌、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氏菌已被证明可以调节水平基因转移和生物膜形成。我们提出的项目 1 基于对几种重要 Cas 蛋白的成功结构测定以及来自 B. halodurans 的 I-C 型级联复合物的成功重建。在本提案中，我们提出了实验来了解 I-C 型 CRISPR-Cas 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型系统中目标搜索级联复合物的结构-功能，（2）表征级联-蛋白质相互作用Cas3的结构-功能，这是一个重要因素。 (3) 捕获 Cascade-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制多样性。 RNA生物学涉及以顺式方式执行基因调控功能的结构化RNA。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。据估计，它们在原核生物中控制着厚壁菌门中多达 2-4% 的基因，它们几乎完全以顺式方式发挥作用，通常驻留在宿主 mRNA 的 5' 非翻译区 (5'- UTR) 中，并调节基因。表达主要通过提前转录终止或抑制翻译起始的方式，其他调节机制包括控制 mRNA 切割、稳定性和选择性剪接已得到证实，我们确定了核糖开关研究的以下前沿，并相应地调整了我们的工作： 1. 利用核糖开关的新型配体传感策略，其研究可能揭示细菌生理学的新方面（该研究）。项目 2 中的 T 盒核糖开关；2. 更深入地了解构象转换机制（项目 3 中的 yybP-ykoY 孤儿核糖开关）；孤儿核糖开关家族的表征（项目3）；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Mn2+传感器应用）。