Messenger RNA Modifications in Translation Regulation

在细胞RNA中已经鉴定出超过100种类型的化学修饰。高分辨率测序的最新技术进展揭示了真核生物mRNA中的修饰，包括N6-甲基腺苷（m6 A）、5-甲基胞嘧啶（m5 C）、其氧化产物5-羟甲基胞嘧啶（hm 5C）、N1-甲基腺苷（m1A）、N6，2 '-O-二甲基腺苷（m6 Am）和假尿苷（pseudouridine，pD）。mRNA修饰可以作为调节特定mRNA翻译的表观遗传标记，然而mRNA修饰对翻译的确切影响知之甚少。本研究的主要目的是阐明m6 A，m5 C和m1A在mRNA上的不同位置时如何调节翻译的不同步骤-起始，延伸，终止和核糖体再循环。为此，我们将使用一系列化学合成的mRNA，在不同的位置进行修饰，并监测它们在由纯化的tRNA、翻译因子和核糖体亚基重建的无细胞酵母翻译系统中的翻译。蛋白质合成的部分反应将利用已建立的生物化学和生物物理方法的工具箱进行真实的实时监测-包括与HPLC偶联的淬灭流的快速动力学和监测荧光和FRET的整体动力学。此外，与卡塞尔大学合作，我们的目标是研究5-甲氧基羰基甲基-2-硫代尿苷（mcm 5s 2 U34）和5-甲基胞嘧啶（m5 C）修饰酵母tRNALys的翻译的各个阶段的协同效应。总之，该项目将提供一个全面的洞察mRNA和tRNA修饰在翻译中的作用。

Over 100 types of chemical modifications have been identified in cellular RNAs. Recent technical advances in high-resolution sequencing have revealed modifications in eukaryotic mRNAs, including N6-methyl adenosine (m6A), 5-methylcytosine (m5C), its oxidation product 5-hydroxymethylcytosine (hm5C), N1-methyladenosine (m1A), N6,2’-O-dimethyladenosine (m6Am) and pseudouridine (Ψ). mRNA modifications may act as epigenetic markers that regulate translation of particular mRNAs, however the exact effect of mRNA modifications on translation in poorly understood. The principle aim of this study is to clarify how m6A, m5C and m1A when present at different locations on mRNA can regulate different steps of translation – initiation, elongation, termination, and ribosome recycling. To this end, we will use array of chemically synthesized mRNAs with modifications at different locations and monitor their translation in a cell-free yeast translation system reconstituted from purified tRNAs, translation factors and ribosomal subunits. The partial reaction of protein synthesis will be monitored in real time utilizing a toolbox of established biochemical and biophysical approaches – including rapid kinetics with quench flow coupled to HPLC and ensemble kinetics monitoring fluorescence and FRET. In addition, in collaboration with the University of Kassel, we aim to study the synergistic effects of the 5-methyloxycarbonylmethyl-2-thiouridine (mcm5s2U34) and 5-methyl-cytosine (m5C) modifications in yeast tRNALys on various stages of translation. Taken together, this project will provide a comprehensive insight into the role of mRNA and tRNA modifications in translation.