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

二氢尿苷(D)是一种转录后RNA修饰，在tRNA中频繁出现，与D-环同名，其结构在二氢尿苷合成酶(DUS)还原某些尿苷残基后变得更加灵活。然而，除了生长表型之外，D的生物学仍然令人惊讶地知之甚少。在这个项目中，申请者展示并寻求现代RNA修饰研究的一个及时特征的令人兴奋的证据，即细胞内D-水平的动力学与酶修饰反应的可逆性相耦合。一个由四个著名的RNA修饰实验室组成的联盟已经产生了两个关键发现，这些发现在这里作为初步数据提供，并形成了一个有趣的工作假说的基础。一方面，tRNA支架中的D残基在酶的作用下被还原为未修饰的尿氨酸，这一点无可辩驳地表明，D作为表位转录标志可以被负责其生物发生的同一种酶去除。另一方面，体内数据显示，用百草枯处理的细胞中D-水平降低，百草枯是一种已知的降低细胞内D-合成辅因子NADPH水平的药物。根据这些流动的工作假设，细胞内NADPH/NADP+水平的比率应该控制tRNAs中D的水平，并可能影响翻译。本申请的特点是一个平衡的工作方案，以基于高端分析、机制和结构研究以及对tRNA修饰模式及其在翻译中的参与的体内调查，深刻阐明工作假说。成功地证明工作假说相当于在同一种酶中识别第一个表位转录编写器/橡皮擦对。它在生命科学中的深远影响尤其包括一种新的分子调控原理，在细胞内氧化还原状态和翻译机器的RNA组件之间发挥作用。

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.