Identification of RNA-binding proteins in Pseudomonas aeruginosa

摘要 铜绿假单胞菌是人类重要的条件致病菌。这是主要原因 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 与铜绿假单胞菌致病能力相关的是两种转录后调节因子RsmA的活性 和Hfq，每一个都影响数百种靶mRNA种类的翻译和/或丰度。 然而，除了RsmA和Hfq之外，对P. 铜绿假单胞菌，或它们发挥的潜在调节作用。我们已经发现，在铜绿假单胞菌中， 预测的RBP（包括RsmA）与冷休克蛋白CspD（本身是RBP）在RNA酶中共纯化 敏感的方式，这意味着它们与相同RNA种类的子集共结合，所述RNA种类被 CspD在目标1中，我们将在铜绿假单胞菌中采用这种亲和共捕获方法与四种额外的RBP， 更全面地了解在这种重要的机会致病菌中发现的RBP。在 目的2，我们将集中于一种蛋白质（PhaF），其以RNA酶敏感的方式与CspD共纯化，这是一种已知的方法。 其作用机制知之甚少的调节剂。具体来说，我们建议识别RNA种类， PhaF结合，确定PhaF的不寻常的核酸结合区域是否代表一种新的RNA- 结合决定簇，并更好地定义PhaF调节子。我们的研究将提供一个纲要的限制性商业惯例在P。 铜绿假单胞菌，并有可能阐明一种新的RNA的调节作用和作用机制， 结合蛋白

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.