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

二氢吡啶（Dihydrouridine， D）是tRNA中常见的转录后RNA修饰，与D-loop同名，二氢吡啶合酶（Dihydrouridine synthases, Dus）还原某些尿嘧啶残基后，其结构变得更加灵活。然而，除了生长表型（可能是翻译的一般效应的结果）之外，D的生物学仍然令人惊讶地不为人所知。在这个项目中，申请人展示和追求现代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的发现，在这里作为初步数据提供，它们构成了一个有趣的工作假设的基础。一方面，tRNA支架中的D残基酶还原为未修饰的尿苷的生化证明，这无可辩驳地表明D作为一个表转录组标记可以被负责其生物发生的同一酶去除。另一方面，体内数据显示，使用百草枯处理的细胞中d水平降低。众所周知，百草枯可以降低细胞内NADPH水平，而NADPH是d合成的Dus辅助因子。从这些流动的工作假设，细胞内NADPH/NADP+水平的比例应该控制trna中的D水平，并可能影响翻译。本应用程序的特点是一个平衡的工作计划，基于高端分析，机制和结构调查，以及tRNA修饰模式及其参与翻译的体内调查，对工作假设进行深刻的阐明。成功地证明工作假设将等同于在同一酶中确定第一个表转录组的写入者/擦除者对。它在生命科学中的深远影响包括一个新的分子调控原理，作用于细胞内氧化还原状态和翻译机制的RNA成分之间。

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.