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。这是导致 囊性纤维化患者的发病率和死亡率，也是医院获得性肺炎的主要原因。中环 与铜绿假单胞菌致病能力有关的是两个转录后调控因子rsma的活性。 和Hfq，每一种都影响数百个目标mRNA物种的翻译和/或丰度。 然而，除了RSMA和Hfq外，人们对P. 铜绿假单胞菌，或它们所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 预测的限制性商业惯例(包括RSMA)在RNase中与冷休克蛋白CSPD共同验证，CSPD本身是一个RBP 敏感的方式，意味着它们共同结合到同一RNA物种的子集上，这些子集由 CSPD。在目标1中，我们将在铜绿假单胞菌中使用这种亲和共捕获方法和另外四个限制性商业惯例来 更全面地了解在这种重要的机会性病原体中发现的限制性商业惯例。在……里面 目标2，我们将关注一种蛋白质(PHAF)，它与CSPD以RNase敏感的方式共同纯化，这是一种已知的 其行动机制鲜为人知的监管者。具体地说，我们建议识别RNA物种 决定PHAF的不寻常的核酸结合区是否代表一种新的RNA- 结合决定簇，并更好地定义PHAF调节子。我们的研究将为P. 并有可能阐明一种新的RNA的调节作用和作用机制。 结合蛋白这种有机体中的结合蛋白

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.