Atlas of stress-induced changes in yeast tRNA modification levels

<jats:p> Transfer RNA (tRNA) modifications are essential for translational accuracy and cellular adaptation to environmental changes. While short-term modification dynamics are well documented, the impact of prolonged stress exposure on the global tRNA landscape remains largely unexplored. This study provides the first systematic profiling of tRNA modifications in <jats:italic>Saccharomyces cerevisiae</jats:italic> following long-term exposure to distinct stress types: heat, suboptimal pH, oxidative stress (paraquat and diamide), osmotic stress (NaCl and KCl), and genotoxic stress (MMS). To this end, we used a broad-range UPLC-MS protocol to quantify global changes in tRNA modification and identify stress-specific signatures. The results revealed that long-term stress triggers a global reprogramming of the tRNA epitranscriptome in a stress-specific and time-dependent manner. Importantly, while our findings confirm the previously reported temperature-sensitivity of wobble uridine thiolation, we also identified a complete or partial loss of 5-methoxycarbonylmethyl-2-thiouridine (mcm <jats:sup>5</jats:sup> s <jats:sup>2</jats:sup> U <jats:sub>34</jats:sub> ) modification upon exposure to paraquat and pH stress. Furthermore, this loss of thiolation is accompanied by an accumulation of the non-thiolated 5-methoxycarbonylmethyl (mcm <jats:sup>5</jats:sup> U) precursor, suggesting a stress-dependent impairment of the thiolation pathway. Next, we applied MarathonRT-based tRNA sequencing and showed that these modification changes occur independently of tRNA isoacceptor abundance. To further integrate these results, we devised a modification deviation ( <jats:italic> MD <jats:sub>m</jats:sub> </jats:italic> ) index, which indicates that the observed reprogramming is primarily linked to events that are independent from changes in tRNA abundance. Together, this study provides a comprehensive atlas of tRNA modification dynamics under prolonged stress, addressing a critical gap in our understanding of RNA-based translational control and establishes the <jats:italic> MD <jats:sub>m</jats:sub> </jats:italic> index as a robust quantitative framework to decouple the influence of tRNA abundance from global modification signals, providing a necessary metric for the field to interpret epitranscriptomic reprogramming. </jats:p>