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.

RNA化学物质生物合成和识别中的序列特异性研究 修改 项目总结 化学修饰在细胞RNA中普遍存在于生命的各个领域；它们扩大了化学修饰的范围 RNA的四个天然构建块之外的空间，调节折叠，分子间相互作用涉及 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 被内源性酶逆转，并能对外部信号做出反应，包括代谢信号、营养 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地 调节RNA的化学修饰被发现在生理缺陷或疾病中调节失调，形成 许多令人兴奋的假说的基础，以发现表位转录组在基因表达调控中的作用。 虽然我们对基于表位转录组学的基因表达调控的理解和治疗开发 在不断扩大的过程中，管理这一调控的分子机制仍然知之甚少。一位批评者 表位转录组域中的问题是各种RNA修饰的序列特异性：它们是如何 安装在特定序列位置上，以及它们如何调节特定的蛋白质-RNA识别。正在研究的 化学修饰的内源RNA的序列环境在技术上一直具有挑战性。使用最近的 高通量测序技术的进步，我们现在只能绘制和量化少数 细胞内天然序列环境中的RNA化学修饰(超过150种类型)。飞行员 作图研究揭示了与特定基因发生相关的保守序列元件 生活中不同领域的改变。这种保守的修改发生，而不是 是随机分布的，强烈表明有专门的内生机制参与其中 调节具有高度特异性的修饰。然而，我们不了解为什么以及如何进行修改 安装在特定位置并调节生物学，很可能是以依赖于序列的方式。致信地址 这一知识缺口，我们需要方法来在序列上下文中自信地检测RNA修改， 提供高精度和高吞吐量。在这里，我们提出了一个计划，重点是开发这种方法和 通过结合进行效应蛋白序列特异性的系统生化表征 体外高通量检测和纤维体块平行报告检测方法。灵感来自于最近 修饰阅读器蛋白质可能识别不止一种化学修饰的发现，我们的目标是重新审视 人类细胞中修饰最多的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和 设计在体外或在细胞报告实验中修改的寡核苷酸文库，我们将阐明 表位转录组关键调控因子的基本生化特性 效率和全面性，导致对RNA调控的更好理解，以及新的机遇 利用和控制表位编码组。