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

研究 RNA 化学修饰生物合成和识别中的序列特异性

• 批准号: 10714628
• 负责人: Huiqing Zhou
• 金额: $ 39.13万
• 依托单位: BOSTON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2028-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10714628
• 关键词: Address; Anabolism; Biochemical; Biological; Biological Assay; Biology; Cells; Chemicals; Conserved Sequence; Cues; Dedications; Defect; Detection; Development; Disease; Elements; Enzymes; Gene Expression; Gene Expression Regulation; High-Throughput Nucleotide Sequencing; Human; In Vitro; Knowledge; Learning; Libraries; Life; Location; Malignant Neoplasms; Maps; Memory; Metabolic; Methods; Modification; Molecular; Nutrient; Oligonucleotides; Oxidative Stress; Physiological; Physiology; Play; Property; Proteins; RNA; RNA Biochemistry; Reader; Regulation; Reporter; Research; Role; Shapes; Signal Transduction; Specificity; Starvation; Technology; Temperature; Therapeutic; Transfer RNA; Visit; design; epitranscriptome; high throughput screening; intermolecular interaction; molecular recognition; programs; tool

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.

研究 RNA 化学物质生物合成和识别中的序列特异性 修改 项目概要 化学修饰普遍存在于生命各个领域的细胞 RNA 中；他们扩大了化学 RNA 的四个天然构建块之外的空间，调节折叠，涉及分子间相互作用 RNA，并调节基因表达。越来越多的证据表明，一些 RNA 化学修饰可以 被内源性酶逆转，并且可以响应外部信号，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，许多蛋白质已知直接或间接 调节RNA的化学修饰被发现在生理缺陷或疾病中失调，形成 发现表观转录组在基因表达调控中的作用的许多令人兴奋的假设的基础。 虽然我们对基于表观转录组的基因表达控制的理解和治疗开发 继续扩大，但控制这种调节的分子机制仍然知之甚少。一个批评的 表观转录组领域的问题是各种 RNA 修饰的序列特异性：它们是如何产生的 安装在特定序列位置以及它们如何调节特定蛋白质-RNA 识别。正在学习 化学修饰的内源 RNA 的序列背景在技术上一直具有挑战性。随着最近 随着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 RNA 化学修饰（超过 150 种类型）在细胞内的天然序列环境中。飞行员 作图研究揭示了与特定事件发生相关的保守序列元件 生活不同领域的改变。这种保守的修饰发生，而不是 是随机分布的，强烈表明专用内源性机制的参与 以高度特异性调节修饰。然而，我们不明白为什么以及如何进行修改 安装在特定位置并调节生物学，很可能以序列依赖的方式。致地址 为了弥补这一知识差距，我们需要在序列背景下自信地检测 RNA 修饰的方法， 提供高精度和吞吐量。在这里，我们提出了一个专注于开发此类方法的计划， 通过结合对效应蛋白的序列特异性进行系统的生化表征 体外高通量测定和细胞内大规模平行报告测定方法。受到最近的启发 研究发现修饰阅读蛋白可能识别不止一种化学修饰，我们的目标是重新审视 人类细胞中修饰最严重的 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 化学修饰图谱， 设计在体外或在细胞报告测定中修饰的寡核苷酸文库，我们将阐明 表观转录组关键调节因子的基本生化特性具有前所未有的 效率和全面性，使人们更好地了解 RNA 调控，并带来新的机遇 利用和控制表观转录组。