New tools for RNA modification dynamics analysis

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After transcription, RNA is processed and chemically modified. These modifications comprise e.g. methylations and thiolations and some of them serve in fine-tuning of translation. Especially in tRNA, the modification status changes in response to stress. How the cells achieve this adaptation remains elusive.The goal of this project is to study the mechanisms of these adaptation processes. For this, we want to use our unique nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS). With this method we can distinguish the origin of the RNA and observe the fate of the RNA present before the stress and the new RNA that is transcribed during the stress. Thus we can identify which RNAs adapt their modification density to overcome the stress. We plan to use yeast cells and study tRNA and ribosomal RNA (rRNA) by total digest and quantitative mass spectrometry of the nucleosides.In addition, we plan to establish an oligonucleotide based NAIL method. This Oligo-NAIL-MS technique will be comparable to SILAC proteomics in protein research. We want to use this method primarily to study modifications in rRNA and how the modification density changes at a defined location in this RNA during stress.