Abstract
Although the biological importance of post-transcriptional RNA modifications in gene expression is widely appreciated, methods to directly detect their introduction 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 show that modifications are introduced in a defined sequential order, and that the chronology is controlled by cross-talk between modification events. In particular, we show that a strong hierarchy controls the introduction of the T54, Ψ55 and m1A58 modifications in the T-arm, and we 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.
Highlights
The biological importance of post-transcriptional RNA modifications in gene expression is widely appreciated, methods to directly detect their introduction during RNA biosynthesis are rare and do not provide information on the temporal nature of events
We developed an original methodology for monitoring the introduction of modified nucleotides in tRNAs using NMR in a time-resolved fashion
A handful of modification circuits have been reported to date, most probably because their study remains difficult, since monitoring the maturation of tRNA in real-time at a single nucleotide level is technically challenging[18]
Summary
The biological importance of post-transcriptional RNA modifications in gene expression is widely appreciated, methods to directly detect their introduction during RNA biosynthesis are rare and do not provide information on the temporal nature of events. By following the maturation of yeast tRNAPhe with time-resolved NMR measurements, we show that modifications are introduced in a defined sequential order, and that the chronology is controlled by cross-talk between modification events. While a simple model would be that modifications are introduced to tRNA independently, several modification circuits have been identified in which one or more modifications stimulate formation of a subsequent modification[7,14] This obviously drives a defined sequential order in the tRNA modification process. Using continuous NMR measurements to measure a series of snapshots of the tRNA along the maturation route, we observe a sequential order in the introduction of several modifications This suggests that modification circuits could control the tRNAPhe maturation process in yeast. We adopted a reverse genetic approach and investigated the interplay between the different modifications in yeast tRNAPhe with both NMR and mass spectrometry and show that modification circuits identified in the yeast extract on tRNAPhe influence the process of tRNA modification in living cells
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