tRNA molecules are key in translating the genetic information into a protein sequence and critical for all aspects of cellular metabolism. Interestingly, tRNAs carry a plethora of chemical modifications, thought to regulate their function. Many of these modifications are evolutionarily highly conserved, and some linked to human degenerative diseases and cancer. However, we know little about their in vivo function and the mechanisms of how aberrations in tRNA modification lead to cellular phenotypes.Thus, my proposal aims at gaining mechanistic insights into the in vivo function of tRNA modifications by performing a comprehensive systems analysis of tRNA modification pathways in yeast: First, I will identify transcripts, which are abnormally translated in tRNA modification mutants, thus causing the observed phenotypes. Importantly, I will define the codon signatures that are the underlying cause. Second, I will develop a mass spectrometric LC-MS/MS-based workflow, allowing for a quantitative and sequence specific analysis of RNA modifications, which is currently not done in Europe. Third, by combining population analyses and long-term evolution experiments, I will address how tRNA modifications constrain the evolution of genomes and modulate cellular stress responses.To achieve my goals, I will combine novel technologies and genetics in an unprecedented manner to push the limits of our understanding. I will employ ribosome profiling, a novel method, that provides high-resolution snapshots of the translatome. I will use quantitative RNA mass spectrometry to establish the link between tRNA modification and changes in translation. Finally, I will use quantitative protein mass spectrometry to verify the translation effects of tRNA hypomodification at the proteome level.Taken together, my project will for the first time unbiasedly link tRNA modifications and translation in vivo by a systems approach and provide an important service to the European RNA community.