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.

研究生物合成和RNA化学识别的序列特异性 修改 项目摘要 化学修饰在生命的所有领域的细胞RNA中都普遍存在。他们扩大化学物质 超出RNA的四个自然构建块，调节折叠，分子间相互作用的空间 RNA并调节基因表达。越来越多的证据表明，几种RNA化学修饰可以是 通过内源性酶逆转，可以响应外部线索，包括代谢信号，营养素 饥饿，氧化应激和温度变化。有趣的是，许多已知直接或间接已知的蛋白质 发现调节的RNA化学修饰在物理缺陷或疾病中失调，形成 许多令人兴奋的假设的基础，以发现表面转录组在基因表达调节中的作用。 同时我们对基于上转录组的基因表达控制的理解和热剥削 继续扩展，控制该调节的分子机制仍然很少理解。批判 表面参考组领域中的问题是各种RNA修改的序列特异性：它们是如何的 安装在特定序列位置及其如何调节特定蛋白-RNA识别。研究 化学修饰的内源性RNA的序列环境在技术上受到了挑战。最近 基于高通量测序的技术的进步，我们现在只能绘制并量化少数几个 RNA化学修饰（超过150种类型）在细胞内部的天然序列上下文中。飞行员 映射研究表明，构成与特定发生有关的序列元素 跨不同领域的修改。这种保守的修改发生，而不是 被随机分布，强烈建议参与专用的内源性机械的参与 调节具有高特异性的修改。但是，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性方式进行。解决 这个知识差距，我们需要在序列上下文中自信地检测RNA修饰的方法 提供高精度和吞吐量。在这里，我们提出了一个计划，重点是开发此类方法和 通过组合对效应蛋白的序列特异性进行系统的生化特异性 体外高通量测定法和纤维素大规模平行的记者测定方法。受到最近的启发 调整读取器蛋白可能识别多种化学修饰的发现，我们旨在重新审视 人类细胞中最重变的RNA - 转移RNA的分子识别机制。 通过开发更先进的检测工具，以绘制生物学RNA的RNA化学修饰和 设计的寡核苷酸库在体外或细胞报告中修饰，我们将阐明 史无前例的表面转录组的关键调节剂的基本生化特性 效率和全面性，从而更好地了解RNA调节和新机会 利用和控制同意组。