Regulation of Messenger RNA by DEAD-box Proteins

DEAD-box 蛋白对信使 RNA 的调节

• 批准号: 8864308
• 负责人: Chaitanya Jain
• 金额: $ 29.55万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8864308
• 关键词: ATP phosphohydrolase; Affect; Alleles; Binding; Binding Sites; Biological; Biology; Boxing; Cell physiology; Cells; Complex; Data; Defect; Disease; Energy-Generating Resources; Escherichia coli; Escherichia coli Proteins; Genetic; Genetic Transcription; Genome; Goals; High-Throughput Nucleotide Sequencing; In Vitro; Knowledge; Laboratories; Mediating; Messenger RNA; Metabolism; Microarray Analysis; Modeling; Molecular Conformation; Mutation; Organism; Play; Process; Production; Property; Protein Binding; Proteins; Publishing; RNA Conformation; RNA Folding; RNA Stability; Reagent; Regulation; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Scientific Advances and Accomplishments; Secondary to; Site; Structure; Suppressor-Effector T-Lymphocytes; Techniques; Testing; Time; Transcript; Translating; Translations; Work; antibiotic design; antimicrobial; base; crosslink; disease-causing mutation; genetic analysis; genome-wide analysis; helicase; in vivo; insight; mutant; novel; novel strategies; premature; protein function; public health relevance; research study; response; stem; transcription termination

 DESCRIPTION (provided by applicant): DEAD-box proteins (DBPs) are key regulators of RNA structure inside the cell and are generally considered to function as ATP-dependent RNA helicases. Although DBPs are implicated in a wide range of cellular processes, the mechanisms by which these factors function inside the cell are not well understood. The objective of the proposed work is elucidate and characterize the in vivo functions of two DBPs from E. coli, DeaD and SrmB. Published data and preliminary results from our laboratory indicate that both factors participate in multiple, partially overlapping aspects of RNA metabolism in the cell. To define their cellular functions in detail, we propose three specific aims. First, both SrmB and DeaD have been implicated in ribosome assembly. Through the identification of genetic suppressors that alleviate the cellular defects caused by an absence of SrmB, we have identified the ribosomal role of this protein is to stimulate the production of a key ribosomal protein, L13. The experiments proposed in this aim will test the hypothesis that SrmB regulates the structure of the mRNA that encodes L13, and thereby, affects L13 production. We will also analyze genetic suppressors of cells that lack DeaD to elucidate the mechanism by which this DBP regulates ribosome assembly. In the second aim, we will globally analyze the regulation of mRNAs in E. coli. Our preliminary data indicate that an absence of either DBP affects the abundance of hundreds of mRNAs. Studies will be performed to test the hypothesis that DBP-mediated structural changes affect RNA expression at the level of transcription termination, stability or translation. We will also investigate whether ATPase-independent activities of the DBPs, such as the ones we have recently identified, are important for transcript regulation in the cell. In the third aim, we will apply a validated cross-linking approach to identify the binding sies of the DBPs on mRNAs and determine RNA binding motifs. Elucidating the basis for DBP- mediated recognition of RNA is critical to understand the underlying basis for RNA regulation. Collectively, these studies will provide multiple insights into the functions of this important famly of RNA regulators, the basis for their interaction with RNA and the mechanisms through which RNA substrates are regulated.

 描述（由申请人提供）：DEAD-box 蛋白 (DBP) 是细胞内 RNA 结构的关键调节因子，通常被认为具有 ATP 依赖性 RNA 解旋酶的功能，尽管 DBP 涉及广泛的细胞过程，但其机制。这些因子在细胞内的作用尚不清楚。本项工作的目的是阐明和表征来自大肠杆菌的两种 DBP（DeaD 和 SrmB）的体内功能。我们实验室公布的数据和初步结果表明，这两个因子参与细胞中 RNA 代谢的多个、部分重叠的方面。为了详细定义它们的细胞功能，我们提出了三个具体目标：首先，SrmB 和 DeaD 都参与其中。通过鉴定可减轻 SrmB 缺失引起的细胞缺陷的基因抑制因子，我们发现该蛋白的核糖体作用是刺激关键核糖体蛋白 L13 的产生。在此目的中，我们将检验 SrmB 调节编码 L13 的 mRNA 结构，从而影响 L13 产生的假设，我们还将分析缺乏 DeaD 的细胞的遗传抑制因子，以阐明该 DBP 调节核糖体组装的机制。第二个目标是，我们将全面分析大肠杆菌中 mRNA 的调控。我们的初步数据表明，任一 DBP 的缺失都会影响数百种 mRNA 的丰度。 DBP 介导的结构变化在转录终止、稳定性或翻译水平上影响 RNA 表达，我们还将研究 DBP 的 ATP 酶独立活性（例如我们最近发现的活性）对于细胞中的转录调节是否重要。在第三个目标中，我们将应用经过验证的交联方法来识别 DBP 在 mRNA 上的结合基序并确定 RNA 结合基序。阐明 DBP 介导的 RNA 识别的基础对于理解 RNA 的潜在基础至关重要。总的来说，这些研究将为这一重要的 RNA 调节因子家族的功能、它们与 RNA 相互作用的基础以及 RNA 底物的调节机制提供多种见解。