Chemical Approaches to Illuminate the Epitranscriptome

项目摘要/摘要 RNA是DNA和蛋白质之间的中心枢纽，了解它的调控对于 阐明正常和疾病生理学中的基因表达程序。信使RNA的性质 (信使核糖核酸)可以被动态的化学修饰(表位转录组)所调节，这些修饰发生在 在转录方面，如N6-甲基腺苷(M6A)，它调节mRNA的周转、翻译、核 输出，剪接，以及对碱基的其他修改。一个主要的挑战是确定 这些修饰的功能后果，并阐明它们控制的分子机制 信使核糖核酸生物学这项建议试图通过发展和应用化学生物学来填补这一知识空白。 确定介导信使核糖核酸修饰对细胞过程的影响的蛋白质的策略。 表位转录组由RNA修饰酶(编写器和擦除器)形成，并由 修饰特异性RNA结合蛋白(读取器)。表征这些蛋白质-RNA的相互作用对于 了解特定修饰的功能和调节。在此之前，我们开发并应用了 化学蛋白质组学策略分析m6A的结合子。我们发现了新的m6A阅读器以及 优先与未修饰的RNA结合。在这里，我们将询问这些m6A介导的蛋白质-RNA的作用 对细胞内mRNA行为的相互作用，并进一步发展我们的方法来读取另一个 甲基化标记N1-甲基腺苷(M1a)。此外，我们还提出了新的方法来表征 体外和细胞内修饰的RNA-蛋白质相互作用。我们的项目有以下具体目标： 目的1.鉴定和功能询问读取mRNA甲基化标记的蛋白质。我们将重点关注 识别和研究M6A和M1A的读者。 目的2.通过体外筛选的方法，对信使核糖核酸甲基化读取器和擦除器的底物特异性进行分析。 目的3.利用RNA邻近连接技术研究甲基化的信使核糖核酸向细胞应激颗粒的转运 策略。 我们的发现将揭示mRNA甲基化如何调节蛋白质-RNA相互作用以控制基因表达。 这些研究应该会提高我们对基本RNA调控机制的理解，并提供 询问信使核糖核酸修饰功能的强大而通用的策略。

PROJECT SUMMARY/ABSTRACT RNA functions as a central hub between DNA and protein, and understanding its regulation is critical to illuminating gene expression programs in normal and disease physiology. The properties of messenger RNA (mRNA) can be modulated by dynamic chemical modifications (the epitranscriptome) that occur post- transcriptionally, such as N6-methyladenosine (m6A), which regulates mRNA turnover, translation, nuclear export, and splicing, as well as other modifications on the nucleobases. A major challenge is to identify the functional consequences of these modifications and elucidate the molecular mechanisms by which they control mRNA biology. This proposal seeks to fill this knowledge gap by developing and applying chemical biology strategies to characterize proteins that mediate the effects of mRNA modifications on cellular processes. The epitranscriptome is shaped by RNA-modifying enzymes (writers and erasers) and interpreted by modification-specific RNA-binding proteins (readers). Characterizing these protein-RNA interactions is critical for understanding the function and regulation of specific modifications. Previously, we developed and applied a chemical proteomics strategy to profile binders of m6A. We identified new m6A readers as well as proteins that bind preferentially to unmodified RNA. Herein, we will interrogate the role of these m6A-mediated protein-RNA interactions on mRNA behavior in the cell and further develop our approach to profile readers of another methylation mark, N1-methyladenosine (m1A). Additionally, we propose novel methodologies to characterize modified RNA-protein interactions in vitro and in the cell. Our project has the following specific aims: Aim 1. Identify and functionally interrogate proteins that read mRNA methylation marks. We will focus on identifying and studying readers of m6A and m1A. Aim 2. Profile the substrate specificity of mRNA methylation readers and erasers by in vitro selection. Aim 3. Investigate the trafficking of methylated mRNA to cellular stress granules using an RNA proximity ligation strategy. Our findings will reveal how mRNA methylation regulates protein-RNA interactions to control gene expression. These studies should improve our understanding of fundamental RNA regulatory mechanisms and provide powerful and general strategies for interrogating the function of mRNA modifications.