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, m5C, and m2 2G, 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, m5C, and m2 2G, 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.

Elongator, a conserved complex required for wobble uridine modifications in Eukaryotes

“…[26][27][28]32 Recently it was suggested that levels of certain modified nucleosides in tRNA, among them ncm 5 U, mcm 5 U and mcm 5 s 2 U could be altered in response to various stress conditions. 67 However, the levels of the ncm 5 U, mcm 5 U and mcm 5 s 2 U nucleosides were not quantified in individual tRNA isoacceptors rather they were quantified in bulk tRNA. Therefore, it is not possible to distinguish between regulation of modification on individual tRNA isoacceptors or if the levels of the tRNA isoacceptors are altered.…”

The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry

“…The paradigmatic example was the work of Dedon and coworkers, who studied the changes in modified nucleoside abundance for yeast as the organism was subjected to different stress conditions. 21 In this study, a single labeled deoxyadenosine standard was mixed with the sample to allow the authors to normalize the signal response from analysis to analysis and from sample to sample. They then found statistically significant differences in the signal response for 23 modifications between untreated and treated yeast cells.…”

“…In accordance with these finding, knock out mutants of trm4 (catalyzes m 5 C) and trm7 (catalyzes Cm) were found to be less viable after hydrogen peroxide exposure as compared to wild type yeast. 21 The capability to routinely monitor dynamic changes in RNA modification levels 22 enabled Dedon and Begley to propose a regulatory model that correlates such changes with the transcriptional and translational needs of an organism. 23 While an internal standard can provide a means to compare 2 samples, a more accurate determination of the quantity of www.landesbioscience.comindividual nucleosides can be obtained by using an isotope dilution method.…”

m6a RNA Methylation: The Implications for Health and Disease

“…These have allowed a greater parity of the occurrence and fluidity of this epigenetic modification [30]. There is growing evidence that these RNA modifications can respond rapidly to a variety of stimuli, particularly stress [31,32]. Recently, FTO has been further implicated in the complicated control of RNA epigenetics by evidence which suggests it may catalyse further reactions oxidising the N(6)-methyladenosine to form the intermediate product N(6)-hydroxymethyladenosine, which is followed by the formation of the further oxidised product N(6)-formyladenosine [33].…”