RNA Modifications as Biomarkers of Environmental Stress and Inflammation

描述（由申请人提供）：拟议的研究有两个目的：进一步了解转录后tRNA修饰的生物学，并探索tRNA修饰作为导致癌症和其他疾病的慢性炎症病理生理学生物标志物的效用。tRNA中有几十种不同的核碱基结构，但大多数这些修饰的确切功能尚不清楚。我们最近证明了tRNA中修饰尿苷（mcm 5U）的酶催化甲基化在DNA损伤后的密码子特异性翻译控制中起作用，并且我们有初步数据表明mcm 5U和其他tRNA修饰的水平在暴露于各种遗传毒素后发生特异性变化。我们假设酶催化的tRNA修饰的水平和模式可以用作毒物暴露和病理生理学的生物标志物。我们的假设是固有的新建议，酶催化的tRNA修饰是损伤信号通路的动态组成部分。我们将使用生物标志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，并更好地了解催化修饰人类tRNA的时间顺序和蛋白质系统。为了实现我们的目标，我们将在细胞暴露于烷化剂和活性氧和氮物质（RONS）之前和之后，对tRNA种类的谱、tRNA核碱基20修饰和tRNA相关基因的表达进行定量评估。超过23个基因特异性的人类tRNA修饰系统，63种tRNA可区分的阵列技术，和超过25个核碱基修饰的组合潜力将进行评估，以测试我们的建议，tRNA为中心的指数是高度特异性的生物标志物，具有很大的动态范围。此外，tRNA为中心的指数将被用来澄清修饰被纳入tRNA的机制基础，并强调tRNA在细胞对有害刺激和病理生理条件，如炎症的反应中的作用。目的1：建立定量RNA核碱基20修饰谱的方法。该目标的目的是开发用于定量tRNA中酶催化的核碱基修饰的分析方法。我们将使用两种质谱方法来定量RNA修饰：全局或非靶向调查和特定核苷的靶向定量。这里的策略是使用全局方法来定义在细胞应激期间经历定量变化的修饰谱，并将修饰与特定的酶和途径相关联。如果在全球研究中确定了暴露“特征”，则可以通过有针对性的策略以更高的灵敏度量化个体修饰。这些方法将用酵母tRNA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为中心的测量作为细胞中暴露的试剂特异性生物标志物特征。这个基于细胞的目标的目的是应用目标#1的方法来评估作为细胞应激的生物标志物的tRNA，并获得对人类tRNA修饰系统的机理性了解。此外，这些研究将提供一个可以转化为体内研究的tRNA特异性变化库。首先，我们将对人HEK293细胞暴露于DNA损伤剂甲磺酸甲酯（MMS）和过氧化氢（H2O2）后修饰核苷的变化进行详细的剂量反应和时间过程研究。结果将指导后续测量（1）对应于tRNA修饰酶的转录物，（2）单个tRNA物质和（3）tRNA修饰的水平，以确定以tRNA为中心的暴露特征。为了检验我们的假设，并作为目标#3的SJL小鼠炎症研究的基准，我们将量化暴露于四种炎症化学介质（H2O2、一氧化氮、过氧亚硝酸盐和HOCl）的培养SJL细胞系中的tRNA中心指数。在所有研究中，将通过计算挖掘得到的定量数据，以确定每种药物的特异性生物标志物特征。此外，我们将使用我们的生物标志物数据来突出tRNA修饰在损伤信号传导中的作用，并提供对人类tRNA修饰系统的机制见解。目的3：使用小鼠炎症模型证明tRNA修饰具有体内生物标志物潜力。应用于目标#2中的细胞的方法和所得模型现在将转化为一氧化氮过度产生和炎症的SJL小鼠模型。这里的目标是量化对应于tRNA修饰酶的转录物，并表征SJL小鼠中暴露于炎症引起的活性氧和氮物质的组织的tRNA修饰谱。将得到的模式与对照动物进行比较，以检验我们的假设，即以tRNA为中心的测量可用作慢性炎症的高度敏感的体内生物标志物，并且以tRNA为中心的测量可用于评估体内炎症部位发生的化学反应.

DESCRIPTION (provided by applicant): 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 20 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 20 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.