A quantitative systems approach reveals dynamic control of tRNA modifications during cellular stress

Abstract

Decades of study have revealed more than 100 ribonucleoside structures incorporated as post-transcriptional modifications mainly in tRNA and rRNA, yet the larger functional dynamics of this conserved system are unclear. To this end, we developed a highly precise mass spectrometric method to quantify tRNA modifications in Saccharomyces cerevisiae. Our approach revealed several novel biosynthetic pathways for RNA modifications and led to the discovery of signature changes in the spectrum of tRNA modifications in the damage response to mechanistically different toxicants. This is illustrated with the RNA modifications Cm, m(5)C, and m(2) (2)G, which increase following hydrogen peroxide exposure but decrease or are unaffected by exposure to methylmethane sulfonate, arsenite, and hypochlorite. Cytotoxic hypersensitivity to hydrogen peroxide is conferred by loss of enzymes catalyzing the formation of Cm, m(5)C, and m(2) (2)G, which demonstrates that tRNA modifications are critical features of the cellular stress response. The results of our study support a general model of dynamic control of tRNA modifications in cellular response pathways and add to the growing repertoire of mechanisms controlling translational responses in cells.

Figure 1. Total ion chromatogram from LC-MS/MS analysis of yeast tRNA ribonucleosides, as described in Materials and Methods.

Figure 2. Hierarchical cluster analysis of toxicant-induced changes in tRNA modification spectra in wild-type yeast exposed to concentrations of MMS, H₂O₂, NaOCl, and NaAsO₂ producing 20%, 50%, and 80% cytotoxicity.

LC-MS/MS quantification of ribonucleosides was performed as described in Materials and Methods and data expressed as fold-change relative to unexposed controls (Table S2), with hierarchical cluster analysis performed on mean-centered data. The top-left color bar indicates the range of fold-change values.

Figure 3. Principal component analysis (PCA) of changes in the levels of tRNA modifications caused by exposure to MMS, H₂O₂, NaOCl, and NaAsO₂.

Toxicant-induced changes in the relative quantities of 23 tRNA ribonucleoside modifications (Table S2) were subjected to PCA following mean centering and normalization of the fold-change data.

Figure 4. Phenotypic analysis of cytotoxicity induced by MMS, NaOCl, H₂O₂, and NaAsO₂ in yeast mutants lacking trm tRNA methyltransferase and other modification genes.

Data represent mean ± SD for three biological replicates. Asterisks denote values statistically different from unexposed controls by Student's t-test, p<0.05. Associated RNA modifications are listed below each enzyme.

Figure 5. Cluster analysis visualization of changes in the relative levels of tRNA ribonucleoside modifications in mutants lacking ribonucleoside-modifying enzymes.

The ratios of ribonucleoside levels from Table S5 were subjected to hierarchical cluster analysis. Red – increases; green – decreases. The top-left color bar indicates the range of fold-change values.