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中修饰的尿苷（mcm5U）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，我们有初步数据表明mcm5U和其他tRNA修饰的水平在暴露于各种基因毒素后特异性改变。我们假设酶催化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。固有的我们的假设是新的建议，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，并更好地理解催化修饰为人类tRNA的时间顺序和蛋白质系统。为了实现我们的目标，我们将对细胞暴露于烷基化剂和活性氧和氮（RONS）之前和之后的tRNA物种光谱、tRNA核碱基20修饰和tRNA相关基因的表达进行定量评估。我们将评估超过23种人类tRNA修饰系统特异性基因、63种可通过阵列技术区分的tRNA物种和超过25种核碱基修饰的组合潜力，以验证我们的建议，即tRNA中心指数是具有大动态范围的高度特异性生物标志物。此外，以tRNA为中心的指数将用于阐明修饰被纳入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。目标1：发展量化RNA核碱基20修饰谱的方法。本目的目的是开发定量酶催化的tRNA核碱基修饰的分析方法。我们将使用两种质谱方法来量化RNA修饰：全局或非靶向调查和特定核苷的靶向量化。这里的策略是使用全局方法来定义在细胞应激过程中发生定量变化的修饰谱，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对个体变化进行量化。这些方法将在酵母tRNA上进行开发和验证，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细胞暴露的特异性生物标志物特征。这个以细胞为基础的目标是应用目标1的方法来评估tRNA作为细胞应激的生物标志物，并获得人类tRNA修饰系统的机制见解。此外，这些研究将提供一个trna特异性变化库，可以转化为体内研究。首先，我们将对人HEK293细胞暴露于DNA损伤剂甲基磺酸盐（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。该结果将指导后续测量(1)tRNA修饰酶对应的转录本水平，(2)单个tRNA物种，以及(3)tRNA修饰以确定tRNA中心暴露特征。为了验证我们的假设，并作为Aim #3 SJL小鼠炎症研究的基准，我们将在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细胞系中量化以trna为中心的指数。在所有的研究中，所得到的定量数据将被计算挖掘，以识别特定于每种药物的生物标志物特征。此外，我们将利用我们的生物标志物数据来强调tRNA修饰在损伤信号传导中的作用，并为人类tRNA修饰系统提供机制见解。目的3：使用小鼠炎症模型来证明tRNA修饰具有体内生物标志物潜力。应用于Aim #2细胞的方法和由此产生的模型现在将被转化为一氧化氮过量产生和炎症的SJL小鼠模型。这里的目标是量化tRNA修饰酶的转录本，并表征SJL小鼠暴露于炎症引起的活性氧和氮的组织中tRNA修饰的光谱。结果将与对照动物进行比较，以验证我们的假设，即tRNA为中心的测量方法可以作为体内慢性炎症的高度敏感生物标志物，tRNA为中心的测量方法可以用来评估体内炎症部位发生的化学反应。公共卫生相关性：RNA修饰作为环境应激和炎症的生物标志物。这些研究将进一步加深我们对转录后RNA修饰生物学的理解，并探索RNA修饰作为慢性炎症病理生理学生物标志物的效用。拟议研究的长期潜力是使用修饰核苷来预测与暴露和炎症诱发癌症相关的风险和临床结果。

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. PUBLIC HEALTH RELEVANCE: RNA Modifications as Biomarkers of Environmental Stress and Inflammation The proposed studies will be used to further our understanding of the biology of posttranscriptional RNA modifications and to explore the utility of RNA modifications as biomarkers of the pathophysiology of chronic inflammation. The long term potential of the proposed research is the use of modified nucleosides to predict risk and clinical outcomes related to exposures and inflammation induced cancers.