Abstract
Cells respond to environmental changes by altering gene expression at several levels, with translational control mechanisms being poorly understood. Emerging evidence points to complex interactions between tRNA, mRNA and ribosomes that control the rate and fidelity of translation. Contributing to this complexity are ~200 genes encoding tRNAs in humans, dozens of genes encoding ribosomal RNA and proteins, and >100 different ribonucleoside modifications in tRNA and rRNA across all organisms in addition to the canonical A, C, G and U. While RNA modifications are emerging as critical players in tRNA stability, stress response and cell growth, information about the higher-level biological function of ribonucleoside modifications is lacking.
To explore the biological function of RNA modifications, the speaker and his research group recently developed a platform for purification of individual RNA species and quantification of the full set of RNA modifications in an organism by liquid chromatography-coupled mass spectrometry. This platform revealed that the dozens of modified ribonucleosides in tRNA behave as a system, with reprogramming of the modifications in response to different types of cell stress. This behavior was observed across the spectrum of living organisms, including bacteria, yeast and mammalian cells. When cells were exposed to a diverse set of toxicants, such as hydrogen peroxide, peroxynitrite, hypochlorous acid, ionizing radiation and a series of alkylating agents, multivariate statistical analysis revealed dynamic shifts in the population of RNA modifications as part of the response to damage, with signature changes for each agent and for different doses of each agent. Further, cells lacking the enzymes involved in synthesizing the tRNA modifications that changed significantly following toxicant exposure proved to be hypersensitive to cytotoxicity caused by the toxicant. Analysis of tRNA modifications affected by hydrogen peroxide exposure revealed a codon-specific translational bias favoring synthesis of specific ribosomal proteins and other stress response proteins. These results suggest a step-wise mechanism of cell response involving reprogramming of tRNA modifications that leads to reprogramming of ribosome structure in the translational control of cellular stress responses.
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