RNA quality control against oxidative damage

描述(由申请者提供)：这个项目的目标是在RNA损伤和质量控制领域为学生提供研究培训，这是一个未被研究的问题，可能与许多人类疾病有关。RNA是基因表达的核心，它通过指导和调节蛋白质的合成，以及通过构成从氨基酸制造蛋白质的机制来实现。基因表达的稳健性取决于纠正可能影响RNA功能的RNA错误的能力。目前的拨款申请旨在研究活性氧物种(ROS)对RNA的损伤，ROS存在于日常生活中，并在氧化应激(OS)等条件下增加。虽然我们对RNA氧化的了解还处于起步阶段，但已经证明，氧化会导致mRNA和rRNA在翻译过程中功能失调，导致异常蛋白质的合成。氧化的RNA的积累可能对细胞和生物体有害。我们的项目旨在揭示特定清除氧化的RNA的分子机制，并防止RNA氧化对细胞活力的有害影响。我们假设去除氧化RNA的主要机制可能包括三个步骤：(1)含有氧化残基的RNA分子被隔离，这一过程可能使用识别氧化RNA的因子；(2)隔离的氧化RNA分子通过RNA降解活动有效地降解为单核苷酸；以及(3)在转录过程中，生成的氧化单核苷酸被移除或被阻止重新结合成RNA。在这个项目中，我们建议使用大肠杆菌来测试两个特定的目标，大肠杆菌是一种可以获得大量RNA新陈代谢信息的细菌。目的1：验证含有剧毒的氧化核苷酸8-oxo-G的RNA从功能部位被移除并被选择性降解的假设。我们将检测不同RNA组分中8-oxo-G的水平，并确定8-oxo-G从功能RNA到降解产物的消除途径。我们将分析RNA降解和8-oxo-G结合活性在该途径中8-oxo-G RNA特异性识别和去除中的作用。目的：确定多核苷酸磷酸化酶(PNPase)在识别和降解含有8-oxo-G的RNA中的特殊作用。我们将分析8-oxo-G结合活性和RNA降解活性的结构决定因素，并研究它们在OS下RNA质量控制中的独立功能。该培训项目为期3年，有6到8名高中、本科和博士前水平的参与者，有0到3年的先前研究经验。预计参与者将在首席调查员的实验室学习和开展实验研究。高度鼓励受训人员公布有意义的成果，作为项目成果的一部分。 公共卫生相关性：项目描述这个研究培训项目将为受训者提供机会来检验这样一个假设，即活着的有机体投资于纠正RNA错误的特定机制，RNA是我们基因发挥作用的分子。我们将研究细胞处理RNA错误的方式，这些错误是由日常生活中不可避免的氧化副产物引起的。该项目的成果将提高公众对RNA损伤对人类健康的潜在影响的认识，并将阐明我们如何预防相关问题。

DESCRIPTION (provided by applicant): The goal of this project is to provide research training to students in the area of RNA damage and quality control, an understudied problem that is potentially related to many human diseases. RNA is central to gene expression by directing and regulating protein synthesis, as well as by constituting the machinery that makes proteins from amino acids. The robustness of gene expression depends on the ability of correcting errors in RNA that may affect RNA function. The present grant application aims at studying RNA damage by reactive oxygen species (ROS), which occurs in everyday life and increases under conditions such as oxidative stress (OS). Although our understanding of RNA oxidation is at its infancy, it has been demonstrated that oxidation renders mRNA and rRNA dysfunctional during translation, causing the synthesis of aberrant proteins. Accumulation of oxidized RNA is presumably detrimental to cells and organisms. Our project is designed to uncover molecular mechanisms that specifically remove oxidized RNA and prevent the deleterious effect of RNA oxidation on cell viability. We hypothesize that a major mechanism for removing oxidized RNA may follow three steps: (1) RNA molecules containing oxidized residues are sequestered in a process that possibly employs factors that recognize oxidized RNA; (2) the sequestered oxidized RNA molecules are efficiently degraded to mononucleotides by RNA degradation activities; and (3) the resulting oxidized mononucleotides are removed or prevented from being reincorporated into RNA during transcription. In this project, we propose to test two specific aims using Escherichia coli, a bacterium for which a large body of information in RNA metabolism is available. Aim 1: To test the hypothesis that RNA containing the highly toxic oxidized nucleotide 8-oxo-G is removed from functional sites and is selectively degraded. We will examine the levels of 8-oxo-G in various RNA fractions and define the pathway that 8-oxo-G is eliminated from functional RNA to degradation products. We will analyze the roles of RNA degradation and 8-oxo-G binding activities in specific recognition and removal of 8-oxo-G RNA in the pathway. Aim 2: To determine the specific roles of polynucleotide phosphorylase (PNPase) in recognizing and degrading 8-oxo-G containing RNA. We will analyze the structural determinants of the 8-oxo-G binding activity and the RNA degradation activity, and study their independent functions in RNA quality control under OS. This training project will last for 3 years, involving 6 to 8 participants at high school, undergraduate and pre-doctoral levels with 0 to 3 years of prior research experiences. The participants are expected to learn and carry out the experimental research in the laboratory of the principal investigator. The trainees are highly encouraged to publish meaningful results as part of the outcome of the project. PUBLIC HEALTH RELEVANCE: Project Narrative This research training project will provide opportunity for the trainees to test the hypothesis that living organisms invest in specific mechanisms for correcting errors in RNA, the molecules through which our genes work. We will study the way cells handle RNA errors that are caused by the unavoidable oxidative byproducts of our daily life. The outcome of this project will enhance public awareness of the potential human health effect of RNA damage and will shed light on how we may prevent related problems.