ABSTRACTAlthough the biological importance of post-transcriptional RNA modifications in gene expression is widely appreciated, methods to directly detect the introduction of these modifications during RNA biosynthesis are rare and do not easily provide information on the temporal nature of events. Here we introduce the application of NMR spectroscopy to observe the maturation of tRNAs in cell extracts. By following the maturation of yeast tRNAPhe with time-resolved NMR measurements, we found

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... t modifications are introduced in a defined sequential order, and that the chronology is controlled by cross-talk between modification events. In particular, we uncovered a strong hierarchy in the introduction of the T54, Ψ55 and m1A58 modifications in the T-arm, and demonstrate that the modification circuits identified in yeast extract with NMR also impact the tRNA modification process in living cells. The NMR-based methodology presented here could be adapted to investigate different aspects of tRNA maturation and RNA modifications in general.