Effectors and regulators of end-dependent mRNA degradation in bacteria

描述(申请人提供)：所有细菌都依赖于信使核糖核酸的降解作为调节基因表达和适应不断变化的生存环境的手段。对于一些转录本，快速降解是由5‘端的磷酸化状态控制的，如果单磷酸化，这可以使RNA衰减增加近一个数量级。RppH最近被发现是负责触发5‘端依赖的降解的酶，它通过从初级转录本的5’端去除磷酸盐来产生短暂的5‘单磷酸化衰退中间产物；然而，我们对RppH在细胞RNA代谢的更大背景下的作用的了解仍然相当初级。这项建议的主要目的是以大肠杆菌和枯草芽孢杆菌为模式生物，加深我们对细菌中5‘端依赖的RNA衰退的理解。为了实现这一目标，将建立一个报告系统来检测5‘端依赖的mRNA降解的变化，正向遗传学将被用于：1.确定焦磷水解酶活性的其他来源；以及2.发现RppH的正负调控因子。有希望的候选者将通过生化和分子生物学方法进一步表征，以确定它们对5‘端依赖的RNA衰退的影响范围和机制。这些研究的结果将加深我们对细菌如何利用RNA降解来控制其基因表达谱的理解，并为已知影响细菌发病机制的调控过程提供见解。 公共卫生相关性：RppH是一种细菌蛋白，此前曾被认为对病原体入侵人类细胞具有重要作用。此外，所有细菌--无论是非致病的还是致病的--都依赖于mRNA的降解来调节基因表达和适应环境。这些研究将加强和深化我们对一个基本的细胞过程的理解，从而为我们提供对影响细菌发病机制的分子机制的洞察。

DESCRIPTION (provided by applicant): All bacteria rely on mRNA degradation as a means to regulate gene expression and to adapt to the ever changing milieu in which they live. For a number of transcripts, rapid degradation is governed by the phosphorylation state of the 5' end, which can potentiate RNA decay by nearly an order of magnitude if monophosphorylated. RppH was recently identified as the enzyme responsible for triggering 5' end-dependent degradation by removing phosphates from the 5' end of primary transcripts to produce short-lived 5' monophosphorylated decay intermediates; however, our knowledge of the role of RppH in the larger context of cellular RNA metabolism remains quite rudimentary. The primary purpose of this proposal is to further our understanding of 5' end-dependent RNA decay in bacteria, using Escherichia coli and Bacillus subtilis as model organisms. To achieve this goal, a reporter system will be created to detect changes in 5' end- dependent mRNA degradation, and forward genetics will be used to: 1. identify additional sources of pyrophosphohydrolase activity; and 2. discover positive and negative regulators of RppH. Promising candidates will be further characterized by biochemical and molecular biological assays to determine the scope and mechanism of their influence on 5' end-dependent RNA decay. The results of these studies will enhance our understanding of how bacteria use RNA degradation to control their gene expression profile and provide insights into a regulatory process known to influence bacterial pathogenesis. PUBLIC HEALTH RELEVANCE: RppH is a bacterial protein previously implicated as being important for pathogens to invade human cells. Furthermore, all bacteria-non-pathogenic and pathogenic alike-rely on mRNA degradation to regulate gene expression and to adapt to their surroundings. These studies will enhance and deepen our understanding of a fundamental cellular process, thereby providing insight into a molecular mechanism known to influence bacterial pathogenesis.