INVESTIGATE SEQUENCE SPECIFICITY IN THE BIOSYNTHESIS AND RECOGNITION OF RNA CHEMICAL MODIFICATIONS

RNA化学物质生物合成和识别中的寡核苷酸序列特异性 修改 项目摘要 化学修饰在生命的所有领域的细胞RNA中普遍存在;它们扩展了化学修饰， RNA的四个天然构建模块之外的空间，调节折叠、分子间相互作用，包括 RNA，并调节基因表达。越来越多的证据表明，几种RNA化学修饰可以 通过内源性酶逆转，可以响应外部信号，包括代谢信号，营养物质， 饥饿、氧化应激和温度变化。有趣的是，许多已知的蛋白质直接或间接 调节RNA化学修饰被发现在生理缺陷或疾病中失调， 许多令人兴奋的假说的基础，发现在基因表达调控中的作用的表转录组。 虽然我们对基于表转录组的基因表达控制的理解和治疗利用 尽管这一领域的研究仍在继续扩大，但对这种调控的分子机制仍知之甚少。一个关键 表转录组领域的一个问题是各种RNA修饰的序列特异性：它们是如何 安装在特定的序列位置，以及它们如何调节特定的蛋白质-RNA识别。研究 化学修饰的内源RNA的序列背景在技术上具有挑战性。与最近 尽管基于高通量测序技术的进步，我们现在只能绘制和量化少数几个 RNA化学修饰（超过150种）在细胞内的天然序列环境中。试点 定位研究揭示了与特异性DNA序列发生相关的保守序列元件。 在不同的生命领域中的变化。这种保守的修饰发生，而不是 是随机分布的，强烈表明参与专门的内源性机制， 以高特异性调节修饰。然而，我们不明白为什么以及如何修改 安装在特定位置并调节生物学，最有可能以序列依赖的方式。解决 这一知识缺口，我们需要方法来检测RNA修饰的序列背景下， 提供高精度和吞吐量。在这里，我们提出了一个计划，重点是发展这种方法， 通过结合以下方法对效应蛋白的序列特异性进行系统的生物化学表征： 体外高通量测定法和细胞内大量平行报告子测定法。灵感来自于最近 研究发现，修饰阅读器蛋白可能识别一种以上的化学修饰，我们的目标是重新审视 人类细胞中最严重修饰的RNA转移RNA的分子识别机制。 通过开发更先进的检测工具来绘制生物RNA中的RNA化学修饰， 设计的寡核苷酸文库在体外或细胞报告基因测定中修饰，我们将阐明 表转录组的关键调节因子的基本生化特性， 效率和全面性，从而更好地了解RNA调控，以及新的机会 来利用和控制表转录组

INVESTIGATE SEQUENCE SPECIFICITY IN THE BIOSYNTHESIS AND RECOGNITION OF RNA CHEMICAL MODIFICATIONS PROJECT SUMMARY Chemical modifications are prevalent in the cellular RNA across all domains of life; they expand the chemical space beyond the four natural building blocks of RNA, modulate folding, intermolecular interactions involving RNA, and regulate gene expression. Growing evidence shows that several RNA chemical modifications can be reversed by endogenous enzymes and can respond to external cues, including metabolic signaling, nutrient starvation, oxidative stress, and temperature change. Interestingly, many proteins known to directly or indirectly regulate RNA chemical modifications are found to be dysregulated in physiological defects or diseases, forming the basis of many exciting hypotheses to discover the role of the epitranscriptome in gene expression regulation. While our understanding and therapeutic exploitation of epitranscriptome-based gene expression control continue to expand, the molecular mechanisms that govern this regulation remain poorly understood. A critical question in the epitranscriptome field is the sequence specificity of various RNA modifications: how they are installed on specific sequence locations and how they regulate specific protein-RNA recognition. Studying the sequence contexts of chemically modified endogenous RNA has been technically challenging. With recent advances in high-throughput sequencing-based technologies, we can now map and quantify only a handful of RNA chemical modifications (out of over 150 types) in their native sequence contexts inside cells. The pilot mapping studies revealed conserved sequence elements associated with the occurrence of specific modifications across different domains of life. That such conserved occurrence of modifications, rather than being randomly distributed, strongly suggests the involvement of dedicated endogenous machinery that regulates modifications with high specificity. However, we do not understand why and how the modifications are installed at specific locations and regulate biology, most likely in a sequence-dependent manner. To address this knowledge gap, we need methods to detect RNA modifications confidently within sequence contexts that offer high accuracy and throughput. Here we propose a program focusing on developing such methods and performing systematic biochemical characterization of the sequence specificity of effector proteins by combining in vitro high-throughput assay and in cellulo massive parallel reporter assay approaches. Inspired by recent findings that modification reader proteins may recognize more than one chemical modification, we aim to revisit the molecular recognition mechanism of the most heavily modified RNA – transfer RNAs in human cells. By developing more advanced detection tools in mapping RNA chemical modifications in biological RNA and designed oligonucleotide libraries modified in vitro or in cellular reporter assays, we will elucidate the fundamental biochemical properties of the critical regulators of the epitranscriptome with unprecedented efficiency and comprehensiveness, leading to a better understanding of RNA regulation, and new opportunities to exploit and control the epitranscriptome.