Identification of RNA-binding proteins in Pseudomonas aeruginosa

铜绿假单胞菌中 RNA 结合蛋白的鉴定

• 批准号: 10613590
• 负责人: SIMON L DOVE
• 金额: $ 22.13万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-25 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10613590
• 关键词: Affinity; Affinity Chromatography; Bacteria; Base Pairing; Binding; Catalogs; Chronic; Disease; Epitopes; Gene Expression; Lung infections; Mediating; Messenger RNA; Molecular Chaperones; Morbidity - disease rate; Nosocomial pneumonia; Nucleic Acid Binding; Organism; Patients; Play; Proteins; Pseudomonas aeruginosa; RNA; RNA Binding; RNA Recognition Motif; RNA-Binding Proteins; Regulon; Ribonucleases; Role; Shock; Transcript; Translations; Virulence; Work; acute infection; chronic infection; cold shock protein; cystic fibrosis patients; experimental study; human pathogen; lung pathogen; mortality; novel; opportunistic pathogen; posttranscriptional; ventilator-associated pneumonia

Abstract Pseudomonas aeruginosa is an important opportunistic pathogen of humans. It is the principal cause of morbidity and mortality in Cystic Fibrosis patients and a major cause of hospital-acquired pneumonia. Central to the ability of P. aeruginosa to cause disease are the activities of two post-transcriptional regulators RsmA and Hfq, each of which influences the translation and/or abundance of hundreds of target mRNA species. However, apart from RsmA and Hfq, little is known about the repertoire of RNA-binding proteins (RBPs) in P. aeruginosa, or the potential regulatory roles they play. We have found that in P. aeruginosa many putative or predicted RBPs (including RsmA) copurify with the cold shock protein CspD, itself an RBP, in an RNase sensitive manner, implying that they are co-bound to subsets of the same RNA species that are bound by CspD. In Aim 1, we will employ this affinity co-capture approach with four additional RBPs in P. aeruginosa to obtain a more comprehensive picture of the RBPs that are found in this important opportunistic pathogen. In Aim 2, we will focus on one protein (PhaF) that co-purifies with CspD in an RNase-sensitive manner, a known regulator whose mechanism of action is poorly understood. Specifically, we propose to identify RNA species that PhaF binds, determine whether the unusual nucleic acid binding region of PhaF represents a novel RNA- binding determinant, and better define the PhaF regulon. Our studies will provide a compendium of RBPs in P. aeruginosa and have the potential to illuminate the regulatory roles and mechanism of action of a novel RNA- binding protein in this organism.

抽象的 铜绿假单胞菌是人类的重要机会病原体。这是 囊性纤维化患者的发病率和死亡率以及医院获得性肺炎的主要原因。中央 铜绿假单胞菌引起疾病的能力是两个转录后调节剂RSMA的活性 和HFQ，每个HFQ都会影响数百种目标mRNA物种的翻译和/或丰度。 但是，除了RSMA和HFQ外，对P中的RNA结合蛋白（RBP）的曲目知之甚少。 Aeruginosa或他们扮演的潜在监管角色。我们发现在铜绿假单胞菌中，许多推定或 预测的RBP（包括RSMA）用冷冲击蛋白CSPD（在RNase中）与RNase中的冷冲蛋白CSPD相关 敏感的方式，这意味着它们与受约束的RNA物种的子集共同绑定 CSPD。在AIM 1中，我们将采用这种亲和力共同捕获方法，并在铜绿假单胞菌中使用另外四个RBP 获取在这种重要的机会性病原体中发现的RBP的更全面的图片。在 AIM 2，我们将重点关注一种以RNase敏感的方式与CSPD共纯化的蛋白质（PHAF），一种已知的 调节器的作用机理知之甚少。具体而言，我们建议鉴定RNA物种 PHAF结合，确定PHAF的异常核酸结合区域是否代表了一种新型的RNA- 结合决定因素，并更好地定义PHAF调节量。我们的研究将在P中提供RBP的汇编。 铜绿疾病，有可能阐明新型RNA-的调节作用和作用机理 在该生物体中结合蛋白质。