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

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.