Regulation of Messenger RNA by DEAD-box Proteins

DEAD-box 蛋白对信使 RNA 的调节

• 批准号: 9069024
• 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/9069024
• 关键词: ATP phosphohydrolase; Affect; Alleles; Binding; Binding Proteins; 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; Health; High-Throughput Nucleotide Sequencing; In Vitro; Knowledge; Laboratories; Mediating; Messenger RNA; Metabolism; Microarray Analysis; Modeling; Molecular Conformation; Mutation; Organism; Play; Process; Production; Property; 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; research study; response; ribosome profiling; 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）：死亡盒蛋白（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，但这些因子在细胞内发挥作用的机制还不清楚。本研究的目的是阐明和表征大肠杆菌中两种消毒副产物的体内功能。coli、DeaD和SrmB。我们实验室已发表的数据和初步结果表明，这两种因素都参与细胞中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都涉及核糖体组装。通过鉴定减轻由于缺乏SrmB而引起的细胞缺陷的遗传抑制因子，我们已经确定了这种蛋白质的核糖体作用是刺激关键核糖体蛋白L13的产生。在这个目标中提出的实验将测试SrmB调节编码L13的mRNA的结构，从而影响L13生产的假设。我们还将分析缺乏DeaD的细胞的遗传抑制因子，以阐明DBP调节核糖体组装的机制。在第二个目标中，我们将从整体上分析E.杆菌我们的初步数据表明，DBP的缺乏会影响数百种mRNA的丰度。将进行研究以检验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表达的假设。我们还将调查是否ATP酶独立活动的DBPs，如我们最近发现的，是重要的转录调控细胞。在第三个目标中，我们将应用一种经过验证的交联方法来鉴定DBP对mRNA的结合位点，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调控的潜在基础至关重要。总的来说，这些研究将提供多种见解的功能，这一重要的家族的RNA调节剂，其相互作用的基础与RNA和RNA底物的调节机制。