Translational regulation in exposure biology: Xenobiotic-induced reprograming oftRNA modifications and selective translation of codon-biased response genes in rat and humanmodels

 描述(申请人提供)：本项目的目标是探索一种新的系统水平的机制，通过调节翻译来使细胞对压力和暴露做出反应。细胞通过信号转导、转录调控和蛋白2°修饰将外部刺激与细胞表型的变化联系起来，从而对异物暴露做出反应。利用一个独特的计算和分析平台，我们最近在酵母中发现了一种新的翻译控制细胞应激反应的机制，在这种机制中，毒物诱导tRNA中数十个修饰的核糖核苷重新编程，通过促进代表应激反应基因家族的密码子偏向mRNAs的选择性翻译来调节蛋白质水平。这项研究的目的是检验一种假说，即通过数十种tRNA修饰中的应激特异性协调变化对基因表达的翻译调控在哺乳动物细胞和组织对异物暴露的反应中发挥作用。大量的大鼠和小鼠模型的初步数据，包括来自国家毒理学计划的DrugMatrix的组织，支持这一假说，强有力的证据表明，异物诱导的tRNA修饰的变化，以及修改tRNA的酶，是复杂组织中应激反应的重要调节因子。在这里，我们将使用人类细胞和大鼠暴露模型来坚定地将tRNA重新编程作为对外源物质反应的翻译调节。具体地说，我们假设暴露于外源化合物促进了tRNA修饰模式中毒物特异性的变化，并且重新编程的tRNA通过选择性翻译偏向密码子的应激反应转录本来调节蛋白质水平。我们将在两个目标上检验这些假设。目的1着重于体外分析与我们发表的酵母暴露结果重叠的烷化和氧化剂电池对人肺和肝细胞tRNA重编程和蛋白质组的变化。这提供了酵母和哺乳动物细胞反应的系统比较。目的2将AIM 1的体外研究转移到体内环境，以验证翻译反应机制发生在复杂组织中的假设。我们首先使用NTP DrugMatrix的组织对大鼠暴露于亚砷酸盐进行了初步研究，以确定tRNA重新编程和iTRAQ蛋白质组学的密码子偏向翻译之间的联系。然后，我们与Melanie Doyle-Eisele博士和Ben Moeller博士(Lovelace Resp)合作，在吸入甲醛的大鼠中测试了翻译控制模型。Res.Inst.)和吉姆·斯温伯格(北卡罗来纳大学)。我们之前的研究表明，肺暴露于甲醛会在鼻腔上皮细胞产生蛋白质和DNA加合物，但不会在更远的肺和肝脏组织中产生蛋白质和DNA加合物，因此我们预计会在鼻腔组织中看到甲醛诱导的tRNA重编程和密码子偏向翻译，而不是肺或肝脏，鼻腔组织与Aim 1的体外研究相匹配。这些研究将为细胞对异种生物应激的一种新的反应机制提供关键的见解，与人类暴露直接相关。未来的研究将把这一模型转化为人类接触药物和炎症性疾病。

 DESCRIPTION (provided by applicant): The goal of this project is to explore a new systems-level mechanism by which cells respond to stresses and exposures by regulating translation. Cells respond to xenobiotic exposures by linking external stimuli to changes in cell phenotype through signal transduction, transcriptional regulation, and protein 2° modifications. Using a unique computational and analytical platform, we recently discovered a new mechanism of translational control of the cell stress response in yeast, in which toxicant-induced reprogramming of dozens of modified ribonucleosides in tRNA regulates protein levels by promoting the selective translation of codon-biased mRNAs representing families of stress-response genes. The goal of the proposed studies is to test the hypothesis that translational regulation of gene expression by stress-specific coordinated changes in the dozens of tRNA modifications plays a role in the response of cells and tissues to xenobiotic exposure in mammals. Abundant preliminary data in rat and mouse models, including tissue from the National Toxicology Program's DrugMatrix, supports this hypothesis, with strong evidence that xenobiotic- induced changes in tRNA modifications, as well as enzymes that modify tRNA, are essential regulators of stress responses in complex tissues. Here we will use human cell and rat exposure models to firmly place tRNA reprogramming as a translational regulator of the response to xenobiotics. Specifically, we hypothesize that exposure to xenobiotics promotes toxicant-specific changes in tRNA modification patterns, and that the reprogrammed tRNAs regulate protein levels by way of selective translation of codon-biased stress response transcripts. We will test these hypotheses in two aims. Aim 1 focuses on in vitro analysis of tRNA reprogramming and proteomic changes in human lung and liver cells exposed to a battery of alkylating and oxidizing agents that overlap with our published yeast exposure results. This provides a systematic comparison of yeast and mammalian cell responses. Aim 2 moves the in vitro studies of Aim 1 to the in vivo setting to test the hypothesis that the translational respons mechanism occurs in complex tissues. We first build on preliminary studies of arsenite exposure in rats using tissues from the NTP DrugMatrix to define the link between tRNA reprogramming and codon-biased translation with iTRAQ proteomics. We then test the translational control model in rats exposed to inhaled formaldehyde in collaboration with Drs. Melanie Doyle- Eisele and Ben Moeller (Lovelace Resp. Res. Inst.) and Jim Swenberg (UNC). We previously showed that pulmonary exposure to formaldehyde generates protein and DNA adducts in nasal epithelium but not more distant lung and liver tissues, so we anticipate seeing formaldehyde-induced tRNA reprogramming and codon- biased translation in nasal tissue but not lung or liver, with nasal tissue matching the in vitro studies of Aim 1. These studies will provide critical insights into a novel mechanism of cell response to xenobiotic stress with direct relevance to human exposures. Future studies will translate this model to human drug exposures and inflammatory diseases.