Expression génétique microbienne, UMR 8261, CNRS, Université de Paris, Institut de biologie physico-chimique, 13 rue Pierre et Marie Curie, 75005, Paris, France.
Department of Chemistry, Ludwig Maximilians University Munich, Butenandtstr. 5-13, 81377, Munich, Germany.
Nat Commun. 2019 Jul 29;10(1):3373. doi: 10.1038/s41467-019-11356-w.
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 tRNA 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 mA58 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.
虽然在基因表达中转录后 RNA 修饰的生物学重要性已被广泛认识，但直接检测其在 RNA 生物合成过程中引入的方法却很少，并且不容易提供有关事件时间性质的信息。在这里，我们介绍了将 NMR 光谱学应用于观察细胞提取物中 tRNA 成熟的方法。通过对酵母 tRNA 的时间分辨 NMR 测量，我们表明修饰是按定义的顺序引入的，并且这种顺序受修饰事件之间的串扰控制。特别是，我们表明一个强层次结构控制着 T 臂中 T54、Ψ55 和 mA58 修饰的引入，并且我们证明了用 NMR 在酵母提取物中鉴定的修饰回路也会影响活细胞中的 tRNA 修饰过程。本文提出的基于 NMR 的方法可以适应于研究 tRNA 成熟和 RNA 修饰的不同方面。
