A Quantitative Systems Approach Reveals Dynamic Control of tRNA Modifications during Cellular Stress

Clement T Y Chan,Koli Taghizadeh,Peter C Dedon,Michael S Demott,Thomas J Begley,Madhu Dyavaiah,Nancy Maizels

doi:10.1371/journal.pgen.1001247

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.

Highlights

The complexity of the transfer RNA system confers great potential for its use in cellular regulatory programs
There are hundreds of transfer RNA (tRNA)-encoding genes in S. cerevisiae and human genomes, with extensive post-transcriptional processing that includes enzyme-mediated ribonucleoside modifications [1]. Considering both tRNA and ribosomal RNA, there are more than 100 known ribonucleoside modifications across all organisms in addition to the canonical adenosine, guanosine, cytidine and uridine [2,3]
To assess the dynamic nature of tRNA modifications proposed by this model, we developed a systems-oriented approach using liquid chromatography-coupled, tandem quadrupole mass spectrometry (LC-MS/MS) to quantify the full set of tRNA modifications in an organism

Summary

Introduction

The complexity of the transfer RNA (tRNA) system confers great potential for its use in cellular regulatory programs. There are hundreds of tRNA-encoding genes in S. cerevisiae and human genomes, with extensive post-transcriptional processing that includes enzyme-mediated ribonucleoside modifications [1] Considering both tRNA and ribosomal RNA (rRNA), there are more than 100 known ribonucleoside modifications across all organisms in addition to the canonical adenosine, guanosine, cytidine and uridine [2,3]. Trm catalyzes the methyl esterification of the uracil-based cm5U and cm5s2U to mcm5U and mcm5s2U, respectively, at the wobble bases of tRNAUCU-ARG and tRNACCU-GLU, among others [17] These wobble base modifications in the tRNA enhance binding of the anticodon with specific codons in mixed codon boxes [18]. Mass spectrometry-based methods have recently emerged as powerful tools for identifying and

Author Summary

Findings

Materials and Methods