Translational regulation in exposure biology - Xenobiotic-induced reprograming of tRNA modifications and selective translation of codon-biased response genes in rat and human models

摘要 人类细胞通过改变基因表达对外源性暴露做出反应，约有50种RNA修饰 包括作为应激反应的关键调节物出现的表转录组。我们开发了独特的 RNA修饰检测（LC-MS/MS，AQRNA-seq）、tRNA基因表达（AQRNA-seq）和计算 （密码子分析）技术，以显示酵母，大鼠肝脏，小鼠和培养的人类细胞响应 外源性暴露与tRNA修饰和tRNA水平的协调变化， 调节重要应激反应蛋白的密码子偏向翻译。用老鼠的肝脏 在NTP DrugMatrix程序中，我们观察到23种不同的tRNA修饰， 在接触后以毒性、剂量和时间依赖性方式发生独特变化。在变化中，亚砷酸盐 增加了肌苷（Q）的水平，这是一种关键的tRNA摆动修饰，可以解码四个氨基酸的密码子。 氨基酸（His、Tyr、Asn、Asp）。这种行为在人肝细胞（HEPG 2）中重现，Q掺入 转化为tRNA以响应细胞活力、活性氧（ROS）解毒所需的亚砷酸钠， 和线粒体功能，并降低Q水平，促进翻译的变化。同样，摆动U tRNA摆动碱基5-甲氧羰基-甲基尿苷（mcm 5 U）也在钠 砷在大鼠肝脏和HepG 2细胞中的作用，敲低相应的摆动U写入器-ALKBH 8- 促进细胞活力降低、ROS增加和线粒体功能改变。我们进一步 表明ALKBH 8缺陷破坏了含硒代半胱氨酸（Sec）的谷胱甘肽过氧化物酶的翻译 GPX是一种解毒ROS并促进线粒体功能的蛋白质。在此更新应用程序中，我们将测试 大鼠和人类细胞中器官特异性tRNA重编程和密码子偏向性翻译假说 暴露在亚砷酸盐和其他有毒物质中，然后测试全血和白色血细胞 白细胞（WBC）用作人表转录组生物标志物研究的可接近的取样室。

ABSTRACT Human cells respond to xenobiotic exposures by altering gene expression, with the ~50 RNA modifications comprising the epitranscriptome emerging as key regulators of the stress response. We have developed unique RNA modification detection (LC-MS/MS, AQRNA-seq), tRNA gene expression (AQRNA-seq), and computational (Codon Analytics) technologies to show that yeast, rat livers, mice and cultured human cells respond to xenobiotic exposures with coordinated changes in the levels of tRNA modifications and tRNAs, to regulate codon-biased translation of important stress response proteins. Using liver from rats exposed to six drugs and toxicants in the NTP DrugMatrix program, we observed that 23 different tRNA modifications were uniquely altered in toxicant-, dose- and time-dependent manner after exposure. Among the changes, arsenite increased the level of queuosine (Q), a key tRNA wobble modification that decodes codons for four amino acids (His, Tyr, Asn, Asp). This behavior was recapitulated in human liver cells (HEPG2), with Q incorporation into tRNA in response to sodium arsenite required for cell viability, reactive oxygen species (ROS) detoxification, and mitochondrial function, and decreased Q levels promoting changes in translation. Similarly, the wobble U tRNA wobble base 5-methoxycarbonyl-methyluridine (mcm5U) was also increased in response to sodium arsenite in rat liver and HepG2 cells, with knockdown of the corresponding wobble U writer – ALKBH8 – promoting decreased cell viability, increased ROS, and changes in mitochondrial function. We have further shown that ALKBH8 defects disrupt the translation of selenocysteine (Sec)-containing glutathione peroxidases (GPXs) that detoxify ROS and promote mitochondrial function. In this renewal application, we will test the hypothesis of organ-specific tRNA reprogramming and codon-biased translation in rats and human cells exposed to arsenite and other toxicants, and then test the idea that whole blood and white blood cells (WBCs) serve as an accessible sampling compartment for human epitranscriptome biomarker studies.