Dihydrouridine RNA modification marks: written and erased by the same enzyme

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Dihydrouridine (D) is a post-transcriptional RNA modification that is frequent in tRNA as the namesake of the D-loop, whose structure becomes more flexible upon reduction of certain uridine residues by dihydrouridine synthases (Dus). However, beyond a growth phenotype, presumably a consequence of a generic effect on translation, the biology of D has remained surprisingly ill understood. In this project, the applicants display and pursue exciting evidence for a timely feature of modern RNA modification research, namely dynamics of intracellular D-levels coupled to reversibility of the enzymatic modification reaction. A consortium of four reputed RNA modification labs has produced two key findings, which, are being supplied here as preliminary data, and which form the basis for an intriguing working hypothesis. There is, on one hand, the biochemical demonstration of enzymatic reversion of D residues in a tRNA scaffold to the unmodified uridines, which irrefutably show that D as an epitranscriptomic mark can be removed by the same enzyme that is responsible for its biogenesis. On the other hand, in vivo data show a reduction of the D-levels in cells treated with a paraquat, an agent known to reduce intracellular levels of NADPH, the Dus cofactor for D-synthesis. From these flows the working hypothesis, that the intracellular ratio of NADPH/NADP+ level should govern the level of D in tRNAs, and potentially affect translation. The present application features a balanced work programme for a profound elucidation of the working hypothesis based on high-end analytics, mechanistic and structural investigations, as well as an in vivo investigations of tRNA modification patterns and their participation in translation. Successfully proving the working hypothesis will be equivalent to identifying the first epitranscriptomic writer/eraser pair in the same enzyme. Its far-reaching consequences in the life sciences include in particular a new principle of molecular regulation, acting between intracellular redox status and RNA components of the translation machinery.