RNA Modifications as Biomarkers of Environmental Stress and Inflammation

A.具体目标 拟议的研究有两个目的：加深我们对转录后生物学的理解 目的：探讨tRNA修饰作为心肌梗死病理生理生物标志物的应用价值。 导致癌症和其他疾病的慢性炎症。有几十个不同的核酸库 TRNA中的结构，但大多数这些修饰的确切功能尚不清楚。我们最近做了 证实了tRNA中修饰的尿苷(Mcm5U)的酶催化甲基化在密码子突变中起作用。 DNA损伤后的特定翻译控制，我们有初步数据表明， Mcm5U和其他tRNA修饰在暴露于各种基因毒素后发生特殊变化。我们 假设酶催化的tRNA修饰的水平和模式可用于 毒物暴露和病理生理学的生物标记物。我们的假设所固有的是小说 提出酶催化的tRNA修饰是损伤信号的动态组成部分 小路。我们将使用生物标记物研究来验证我们的假设，建立酶催化的tRNA 修饰作为损伤信号通路的组成部分，并更好地理解时间顺序和 催化修饰成人类tRNA的蛋白质系统。为了实现我们的目标，我们将履行 TRNA物种谱的定量评估、tRNA核酸基2修饰和 细胞暴露于烷化剂和活性氧前后tRNA相关基因的表达 和氮物种(RON)。23种以上人类tRNA特异性基因的结合潜能 修饰系统，63种可通过阵列技术区分的tRNA物种，以及超过25个碱基对 将对修改进行评估，以测试我们的建议，即以tRNA为中心的指数是具有高度特异性的生物标志物 较大的动态范围。此外，将使用以tRNA为中心的指数来阐明 将修饰结合到tRNA中，以突出tRNA在细胞对有害物质的反应中的作用 刺激和病理生理条件，如炎症。 目的1：建立定量测定核糖核酸碱基修饰谱的方法。目标是 这一目标的目的是开发分析方法来量化酶催化的碱基修饰 TRNA。我们将使用两种质谱学方法来量化RNA修饰：全局的或非靶向的 对特定核苷进行调查和有针对性的量化。这里的战略是使用全球方法来 定义在细胞应激过程中发生数量变化的修饰的谱，并将其关联 用特定的酶和途径进行修饰。如果在全球研究中确定了暴露“签名”， 个别修改可以通过有针对性的策略以更高的敏感度进行量化。这些方法将 用酵母tRNA进行开发和验证，然后应用于小鼠和人类细胞的tRNA。 目的2：评估以tRNA为中心的测量方法作为暴露于 细胞。这个基于细胞的目的是应用目标1的方法来评估tRNA作为生物标记物。 对细胞压力的研究，以及对人类tRNA修饰系统的机械洞察。此外，这些 研究将提供一个tRNA特异性改变的文库，可以转化为体内研究。首先，我们将 进行详细的剂量-反应和时间过程研究，以了解暴露后修饰核苷的变化 甲磺酸甲酯和过氧化氢对人HEK293细胞DNA损伤的影响 (过氧化氢)。这些结果将指导随后对(1)与tRNA相对应的转录本水平的测量 修饰酶，(2)单个tRNA种类，和(3)用于测定tRNA的tRNA修饰。 以曝光为中心的签名。来检验我们的假设，并作为SJL小鼠炎症的基准 研究目标#3，我们将量化暴露于四种化学物质的培养SJL细胞系中的tRNA中心指数 炎症介质(过氧化氢、一氧化氮、过氧亚硝酸盐和高氯酸盐)。在所有研究中，由此产生的量化 数据将被计算挖掘，以识别特定于每种药物的生物标记物签名。此外，我们将使用 我们的生物标记物数据，以突出tRNA修饰在损伤信号中的作用，并提供 洞察人类tRNA修饰系统。 目的3：使用小鼠炎症模型来证明tRNA修饰在体内具有 生物标志物潜力。现在将翻译应用于AIM#2中的单元格的方法和结果模型 给SJL小鼠造成一氧化氮过度产生和炎症的模型。这里的目标是量化 与tRNA修饰酶相对应的转录本，并表征tRNA的谱 暴露于炎症产生的活性氧和氮物种的组织的修饰 SJL小鼠。结果模式将与对照动物进行比较，以检验我们的假设：tRNA- 中心性测量可作为慢性炎症的高度敏感的体内生物标志物，tRNA- 中心性测量可以用来评估体内炎症部位发生的化学物质。

A. SPECIFIC AIMS The proposed studies have two objectives: to further our understanding of the biology of posttranscriptional tRNA modifications and to explore the utility of the tRNA modifications as biomarkers of the pathophysiology of chronic inflammation leading to cancer and other diseases. There are dozens of different nucleobase structures in tRNA, yet the precise function of most of these modifications is unknown. We have recently demonstrated that enzyme-catalyzed methylation of a modified uridine (mcm5U) in tRNA plays a role in codon- specific translational control after DNA damage, and we have preliminary data demonstrating that the levels of mcm5U and other tRNA modifications specifically change after exposure to a variety of genotoxins. We hypothesize that the levels and patterns of enzyme-catalyzed tRNA modifications can be used as biomarkers of toxicant exposures and pathophysiologies. Inherent to our hypothesis is the novel proposal that enzyme-catalyzed tRNA modifications are dynamic components of damage signaling pathways. We will use biomarker studies to test our hypothesis, to establish enzyme-catalyzed tRNA modifications as components of damage signaling pathways, and to better understand the temporal order and protein systems that catalyze modifications into human tRNA. To accomplish our objectives, we will perform quantitative assessments of the spectrum of tRNA species, tRNA nucleobase 2¿ modifications, and the expression of tRNA-related genes, before and after exposure of cells to alkylating agents and reactive oxygen and nitrogen species (RONS). The combinatorial potential of more than 23 genes specific to human tRNA modification systems, 63 tRNA species distinguishable by array techniques, and more than 25 nucleobase modifications will be assessed to test our proposal that tRNA-centric indices are highly specific biomarkers with a large dynamic range. Further, tRNA-centric indices will be used to clarify the mechanistic basis by which modifications are incorporated into tRNA and to highlight the role of tRNA in the cellular response to noxious stimuli and pathophysiological conditions such as inflammation. Aim 1: Develop methods to quantify the spectrum of RNA nucleobase 2¿ modifications. The objective of this aim is to develop analytical methods for quantifying enzyme-catalyzed nucleobase modifications in tRNA. We will use two mass spectrometric approaches to quantify RNA modifications: global or untargeted surveys and targeted quantification of specific nucleosides. The strategy here is to use the global approach to define the spectrum of modifications undergoing quantitative changes during cell stress and to correlate modifications with specific enzymes and pathways. If an exposure "signature" is identified in global studies, the individual modifications can be quantified with greater sensitivity by a targeted strategy. The methods will be developed and validated with yeast tRNA and then applied to tRNA from mouse and human cells. Aim 2: Assessment of tRNA-centric measures as agent-specific biomarker signatures of exposure in cells. The objectives of this cell-based aim are to apply the methods of Aim #1 to assess tRNA as a biomarker of cellular stress and to gain mechanistic insight into human tRNA modification systems. Further, these studies will provide a library of tRNA-specific changes that can be translated into in vivo studies. First, we will perform detailed dose-response and time course studies for changes in modified nucleosides after exposure of human HEK293 cells to the DNA damaging agents methyl methanesulfonate (MMS) and hydrogen peroxide (H2O2). The results will guide subsequent measurements of the levels of (1) transcripts corresponding to tRNA modification enzymes, (2) individual tRNA species, and (3) tRNA modifications for determination of tRNA- centric exposure signatures. To test our hypothesis and as a benchmark for the SJL mouse inflammation studies of Aim #3, we will quantify tRNA-centric indices in cultured SJL cell lines exposed to four chemical mediators of inflammation (H2O2, nitric oxide, peroxynitrite and HOCl). In all studies, the resulting quantitative data will be computationally mined to identify biomarker signatures specific to each agent. Further, we will use our biomarker data to highlight a role for tRNA modifications in damage signaling and to provide mechanistic insight into human tRNA modification systems. Aim 3: Use a mouse model of inflammation to demonstrate that tRNA modifications have in vivo biomarker potential. The methods applied to cells in Aim #2 and the resulting models will now be translated to the SJL mouse model of nitric oxide over-production and inflammation. The goal here is to quantify the transcripts corresponding to tRNA modification enzymes and to characterize the spectrum of tRNA modifications from tissues exposed to the reactive oxygen and nitrogen species arising from inflammation in the SJL mice. The resulting patterns will be compared to control animals to test our hypotheses that tRNA- centric measures can be used as highly sensitive in vivo biomarkers of chronic inflammation and that tRNA- centric measures can be used to assess the chemistry that occurs at sites of inflammation in vivo.