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: 10428914
• 负责人: SIMON L DOVE
• 金额: $ 26.55万
• 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 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10428914
• 关键词: Affinity; Affinity Chromatography; Bacteria; Base Pairing; Binding; Catalogs; Chronic; Disease; Epitopes; Gene Expression; Genetic Transcription; 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; 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重要的条件致病菌。这是造成的主要原因 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与两个转录后调节因子 RsmA 的活性有关 和 Hfq，每一个都会影响数百种目标 mRNA 的翻译和/或丰度。 然而，除了 RsmA 和 Hfq 之外，人们对 P. . 铜绿假单胞菌，或它们发挥的潜在调节作用。我们发现，在铜绿假单胞菌中，许多假定的或 预测的 RBP（包括 RsmA）与冷休克蛋白 CspD（本身就是 RBP）在 RNase 中共纯化 敏感的方式，这意味着它们与相同 RNA 种类的子集共同结合，这些 RNA 种类由 CspD。在目标 1 中，我们将采用这种亲和共捕获方法与铜绿假单胞菌中的四个附加 RBP 来 更全面地了解这种重要的机会性病原体中发现的 RBP。在 目标 2，我们将重点关注一种以 RNase 敏感方式与 CspD 共纯化的蛋白质 (PhaF)，这是一种已知的 其作用机制尚不清楚。具体来说，我们建议鉴定 RNA 种类 PhaF 结合，确定 PhaF 的不寻常核酸结合区域是否代表一种新的 RNA- 结合决定子，并更好地定义 PhaF 调节子。我们的研究将提供 P.RBP 的概要。 铜绿假单胞菌，并有可能阐明新型 RNA 的调节作用和作用机制 该生物体中的结合蛋白。