Innate immune and chemically triggered oxidative stress modifies translational fidelity

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Abstract
For cells to function properly the process of translating RNA messengers into proteins needs to be accurate, on the whole. Yet work in HeLa cells now shows that about 1% of methionine residues used in protein synthesis are aminoacylated to 'textbook-incorrect' tRNAs. Surprisingly, the proportion of Met-misacylated tRNAs increases significantly when cells are under stress through viral infection or treatment with viral or bacterial Toll-like receptor ligands. Tests with other amino acids indicate that the phenomenon is limited to Met, and as Met residues are known to protect proteins against damage from reactive oxygen species, one possibility is that Met-misacylation is a natural protective response to cellular stress. Accurate transfer RNA (tRNA) aminoacylation is necessary for translational fidelity; however, the accuracy of tRNA aminoacylation in vivo is uncertain. In mammalian cells, approximately 1% of methionine residues used in protein synthesis are now shown to be aminoacylated to non-methionyl-tRNAs. Furthermore, misacylation of methionine increases up to tenfold upon exposing cells to viruses, toll-like receptor ligands or oxidative stress. Translational fidelity, essential for protein and cell function, requires accurate transfer RNA (tRNA) aminoacylation. Purified aminoacyl-tRNA synthetases exhibit a fidelity of one error per 10,000 to 100,000 couplings1,2. The accuracy of tRNA aminoacylation in vivo is uncertain, however, and might be considerably lower3,4,5,6. Here we show that in mammalian cells, approximately 1% of methionine (Met) residues used in protein synthesis are aminoacylated to non-methionyl-tRNAs. Remarkably, Met-misacylation increases up to tenfold upon exposing cells to live or non-infectious viruses, toll-like receptor ligands or chemically induced oxidative stress. Met is misacylated to specific non-methionyl-tRNA families, and these Met-misacylated tRNAs are used in translation. Met-misacylation is blocked by an inhibitor of cellular oxidases, implicating reactive oxygen species (ROS) as the misacylation trigger. Among six amino acids tested, tRNA misacylation occurs exclusively with Met. As Met residues are known to protect proteins against ROS-mediated damage7, we propose that Met-misacylation functions adaptively to increase Met incorporation into proteins to protect cells against oxidative stress. In demonstrating an unexpected conditional aspect of decoding mRNA, our findings illustrate the importance of considering alternative iterations of the genetic code.