Messenger RNA Modifications in Translation Regulation

翻译调控中信使 RNA 的修饰

• 批准号: 404931650
• 负责人: Professorin Dr. Marina V. Rodnina, since 4/2021
• 金额: --
• 依托单位: Abteilung Physikalische Biochemie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/404931650?language=en
• 关键词: Messenger; RNA; Modifications; Translation; Regulation

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.

在细胞rna中已经发现了100多种化学修饰。高分辨率测序技术的最新进展揭示了真核mrna的修饰，包括N6-甲基腺苷（m6A）， 5-甲基胞嘧啶（m5C），其氧化产物5-羟甲基胞嘧啶（hm5C）， n1 -甲基腺苷（m1A）， N6,2 ' - o -二甲基腺苷（m6Am）和假尿嘧啶（Ψ）。mRNA修饰可能作为调控特定mRNA翻译的表观遗传标记，然而mRNA修饰对翻译的确切影响尚不清楚。本研究的主要目的是阐明m6A、m5C和m1A在mRNA上的不同位置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，我们将使用一系列在不同位置进行修饰的化学合成mrna，并在由纯化的trna、翻译因子和核糖体亚基重组的无细胞酵母翻译系统中监测它们的翻译。蛋白质合成的部分反应将利用已建立的生化和生物物理方法的工具箱进行实时监测-包括快速动力学与淬灭流耦合高效液相色谱和集合动力学监测荧光和FRET。此外，我们与卡塞尔大学合作，旨在研究酵母tRNALys中5-甲基氧羰基甲基-2-硫吡啶（mcm5s2U34）和5-甲基胞嘧啶（m5C）修饰在翻译不同阶段的协同作用。综上所述，该项目将全面了解mRNA和tRNA修饰在翻译中的作用。