A systems biology approach unravels the biological function of two tRNA wobble base modifications in fine-tuning translation and the response to environmental stress

Abstract

tRNA molecules undergo extensive post-transcriptional modifications to produce several variations of the four canonical nucleotides. Despite the huge number of nucleotide modifications that have been identified in tRNAs, to date their biological roles and regulations continue to be poorly understood. The uridines at the wobble position of the eukaryotic cytoplasmic tRNAs tK^UUU, tQ^UUG and tE^UUC are methoxycarbonylmethylated and thiolated to form mcm⁵s²U₃₄ by the ELP- and URM1-pathways, respectively. Several in vitro experiments have implicated these modifications in modulating wobbling capacity. Moreover, mutations in the ELP- and URM1-pathway genes have been associated with physiological defects in several organisms, including complex neurological disorders in humans. In this thesis we used a systems biology approach to study the in vivo functional relevance of mcm⁵s²U₃₄. A sensitive, robust and quantitative proteomics workflow was developed and applied to investigate differential proteome composition in budding yeast mutants deficient in U₃₄ modifications. We find that, in vivo and under normal conditions, mcm⁵s²U₃₄ fine tunes proteome composition by ensuring efficient translation of mRNAs biased for AAA, CAA and GAA codons. Importantly, our results connect these tRNA modifications with various cellular stress response pathways. Follow up analyses of yeast cells subjected to environmental stresses were conducted and led to the discovery that the biosynthesis of mcm⁵s²U₃₄ is dynamically regulated in response to growth conditions in a URM1-pathway dependent fashion. We propose that this regulation allows the cells to adjust their translational capacity during unfavourable growth conditions and contributes to the management of the environmental stress response.
Overall, this thesis presents the first extensive investigation of the functional relevance of tRNA nucleotide modifications and reports one of the few known cases wherein cells regulate the levels of modified nucleotides to fine tune their metabolism in response to environmental cues. We expect that dynamic modulation of RNA modifications will prove to be a more general regulatory mechanism of cellular processes. The experimental and analytical approaches presented in this dissertation will provide a general framework for future studies in this field.

Date of Award	2014
Original language	English
Supervisor	Patrick Pedrioli (Supervisor)