Dynamics of methylated nucleosides and their derivatives in RNAs

RNA 中甲基化核苷及其衍生物的动态

• 批准号: 277312423
• 负责人: Professor Dr. Markus T. Bohnsack
• 金额: --
• 依托单位: Institut für Organische Chemie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/277312423?language=en
• 关键词: Dynamics; methylated; nucleosides; their; derivatives

Modifications in cellular RNAs, collectively termed the “epitranscriptome”, have recently been shown to be more widespread in cellular RNAs than previously anticipated and they have emerged as critical regulators of most aspects of RNA metabolism. Thereby, they play important roles in modulating fundamental cellular processes such as gene expression, the cell cycle, development and differentiation. Recent advances in next-generation sequencing-based methods for transcriptome-wide mapping of RNA modifications have revealed the presence of pseudouridine, N6-methyladenosine (m6A), N6-2’-O-dimethyladenosine (m6Am), 5-methylcytosine (m5C), 1-methyladenosine (m1A) and 2’-O-methylations (Nm) in messenger RNAs (mRNAs) and many other RNA species. While knowledge of the enzymes responsible for installing specific RNA modifications (“writers”) continues to expand, interestingly, recent work has uncovered the presence of demethylases that can act as modifications “erasers” as well as “reader” proteins that specifically bind modified RNAs thereby regulating their biogenesis, functions and stability. Together with the discovery that many RNA modifications are present at substoichiometric levels, this has highlighted the dynamic nature of the epitranscriptome and emphasised the importance of RNA modifications as key regulators of gene expression. During the first funding period THE project, we have explored the roles of m6A and m5C modifications in RNAs, including the characterisation of various writer and reader proteins as well as the dissection of the interactions these factors establish with their substrates on the molecular and atomic levels. Building on these findings, in this project we will further address the functions, substrate specificity and mode of action of different modification enzymes that contribute to the dynamics of RNA modifications. The cellular RNA-interactomes of such enzymes will be determined using UV crosslinking and analysis of cDNA (CRAC) and other in vivo analyses, and the spectrum of nucleotides targeted by these enzymes will be identified. The development of transcriptome-wide mapping approaches for selected RNA modifications and the analysis of the effects of loss of specific modifications in normal growth conditions and also upon exposure to stresses will provide insight into the functions of RNA modifications in modulating gene expression. Structural and biochemical analyses will be employed to elucidate the fundamental principles of substrate recognition and catalysis by these enzymes.

细胞RNA的修饰，统称为表位转录组，最近被证明在细胞RNA中比以前预期的更广泛，它们已经成为RNA新陈代谢的大多数方面的关键调节因子。因此，它们在调控基因表达、细胞周期、发育和分化等基本细胞过程中发挥着重要作用。新一代基于测序的RNA修饰转录组定位方法的最新进展揭示了信使RNA(MRNAs)和许多其他RNA物种中存在假尿苷、N6-甲基腺苷(M6A)、N6-2‘-O-二甲基腺苷(M6Am)、5-甲基胞嘧啶(M5C)、1-甲基腺苷(M1a)和2’-O-甲基化(Nm)。虽然对负责安装特定RNA修饰的酶(“编写者”)的知识继续扩大，但有趣的是，最近的工作发现了可作为修饰“擦除器”的去甲基酶的存在，以及专门结合修饰RNA的“读取器”蛋白质，从而调节其生物发生、功能和稳定性。再加上许多RNA修饰存在于亚化学计量水平，这突显了表位转录组的动态性质，并强调了RNA修饰作为基因表达的关键调节因子的重要性。在项目的第一个资助期，我们探索了M6A和M5C修饰在RNA中的作用，包括各种写入物和读取物蛋白的特征，以及这些因素在分子和原子水平上与其底物建立的相互作用的剖析。在这些发现的基础上，在这个项目中，我们将进一步研究影响RNA修饰动力学的不同修饰酶的功能、底物特异性和作用模式。这些酶的细胞RNA相互作用将通过紫外光交联和DNA分析(CRAC)和其他体内分析来确定，并将确定这些酶靶向的核苷酸的谱。为选定的RNA修饰开发转录组范围的作图方法，并分析在正常生长条件下以及在胁迫下丢失特定修饰的影响，将为深入了解RNA修饰在调控基因表达中的功能提供帮助。结构和生化分析将被用来阐明底物识别和这些酶催化的基本原理。