Detection and Identification of new RNA modifications

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The central objective of this project is the discovery and identification of hitherto unknown RNA modifications. The detection itself is centered on a combination of liquid chromatography and mass spectrometry (LC-MS) and stable isotope labelling techniques. A so called neutral loss screening (NLS) method, developed for the general detection of all ribose-containing nucleosides, has identified >100 candidate compounds in tRNA preparations from E. coli . For 37 of these high-quality data sets suggest that their structures can be elucidated in short order. As a proof-of-concept, the structure of one candidate, later named msms2i6A, was investigated. We could show that this new modification contains a thioacetal structure, which is generated by the repeated action of the radical-SAM enzyme MiaB on i6A. For the second grant period, we propose to clarify the structures of several more candidates, and to detect new candidate signals in RNA preparations from various origins. The latter may lead to the estimation of a new “ceiling” number of existing RNA structures. Furthermore, the biogenesis of different candidates will be investigated by screening their presence in knockout strains of known modification enzymes. We also expect to gain more insight into the biological relevance of various candidates by analyzing their presence in biologically functional assemblies such as polysomes.