RNA quality control against oxidative damage

描述（由申请人提供）：该项目的目标是为学生提供RNA损伤和质量控制领域的研究培训，这是一个未充分研究的问题，可能与许多人类疾病有关。RNA通过指导和调节蛋白质合成，以及通过构成从氨基酸制造蛋白质的机制，对基因表达至关重要。基因表达的鲁棒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（ROS）对RNA的损伤，这种损伤发生在日常生活中，并在氧化应激（OS）等条件下增加。虽然我们对RNA氧化的理解还处于起步阶段，但已经证明氧化使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异性去除氧化RNA的分子机制，并防止RNA氧化对细胞活力的有害影响。我们推测，去除氧化RNA的主要机制可能遵循三个步骤：（1）含有氧化残基的RNA分子在一个可能利用氧化RNA识别因子的过程中被隔离;（2）被隔离的氧化RNA分子通过RNA降解活性被有效降解为单核苷酸;和（3）在转录过程中除去或防止所得氧化单核苷酸重新掺入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，大肠杆菌是一种细菌，可以获得大量的RNA代谢信息。目标1：检验含有高毒性氧化核苷酸8-oxo-G的RNA从功能位点移除并选择性降解的假设。我们将检查各种RNA组分中8-oxo-G的水平，并确定8-oxo-G从功能RNA中消除为降解产物的途径。我们将分析RNA降解和8-oxo-G结合活性在特异性识别和去除途径中的8-oxo-G RNA中的作用。目的2：研究多核苷酸磷酸化酶（PNIPs）在识别和降解8-oxo-G RNA中的特异性作用。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。该培训项目将持续3年，涉及6至8名具有0至3年研究经验的高中，本科和博士前水平的参与者。参与者将在主要研究者的实验室学习和开展实验研究。大力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：项目叙述本研究培训项目将为学员提供机会，以检验生物体投资于纠正RNA错误的特定机制的假设，RNA是我们基因工作的分子。我们将研究细胞处理RNA错误的方式，这些错误是由我们日常生活中不可避免的氧化副产物引起的。该项目的结果将提高公众对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.