NAIL-MS Elucidates Crucial tRNA U34 Modifications in Response to Heat Stress across Eukaryotes and Prokaryotes

Global warming leads to rising temperatures, necessitating organismal adaptation at the cellular level. One potential mechanism for maintaining proteome integrity during stress is the adaptation of tRNA modifications. While tRNA modification reprogramming has been well-studied under chemical stressors, its role in heat stress remains unclear. To address this, we performed a comparative analysis of tRNA modifications in Arabidopsis thaliana, Saccharomyces cerevisiae, Caenorhabditis elegans, Dictyostelium discoideum, and Escherichia coli under heat stress. We assessed the abundance of 30 modified nucleosides using isotope dilution mass spectrometry under control conditions. A. thaliana showed a similar diversity and abundance of tRNA modifications compared to other eukaryotes, suggesting conservation across species. Under heat stress, overall tRNA modification levels were largely stable, with no significant changes in modifications such as dihydrouridine and N4-acetylcytidine. However, one to four modifications per organism were altered, with uridine modifications at position 34 (U34) being the most prominent change. Here, pulse-chase NAIL-MS (nucleic acid isotope labeling coupled mass spectrometry) experiments in E. coli and S. cerevisiae revealed that changes in U34 modifications occurred not only in pre-existing tRNAs but also in newly transcribed tRNAs. These results suggest that existing tRNAs adapt as an early response to heat stress, while newly transcribed tRNAs are reprogrammed to ensure long-term survival under prolonged heat. Our findings highlight the potential role of tRNA modification reprogramming in heat stress adaptation.