Center for dynamic RNA epitranscriptomes - Renewal

动态 RNA 表观转录组中心 - 更新

• 批准号: 10615783
• 负责人: CHUAN HE
• 金额: $ 250万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-27 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10615783
• 关键词: Adenosine; Affect; Animal Model; Area; Basic Science; Binding Sites; Biological; Biological Process; Biology; Cell Differentiation process; Chemicals; Chemistry; Chromatin; Chromosomes; Clinical; Communities; Complex; Databases; Dedications; Development; Disease; Engineering; Enhancers; Enzymes; Evolution; Gene Expression Regulation; Genetic Transcription; Genomics; Introns; Investigation; Life; Mammals; Maps; Medical; Messenger RNA; Methods; Methylation; Modification; Mutation; Neurons; Nucleic Acids; Physiological; Play; Post-Transcriptional RNA Processing; Property; Proteins; Protocols documentation; Pseudouridine; RNA; RNA Processing; RNA metabolism; RNA-Binding Proteins; Reaction; Reader; Regulation; Research; Resolution; Reverse Transcription; Ribosomal RNA; Role; Sampling; Site; Study models; System; Testing; Tissues; Transcript; Transfer RNA; Translations; Untranslated RNA; Up-Regulation; Work; base; bioinformatics tool; chemical group; computational pipelines; demethylation; epitranscriptome; epitranscriptomics; experimental study; human disease; invention; method development; nanopore; new technology; posttranscriptional; promoter; sequencing platform; stem cell differentiation; transcriptome

Project Summary/Abstract Chemical modifications on mammalian messenger RNA (mRNA) have recently been shown to play critical and diverse regulatory roles in mRNA metabolism and translation. For example, the most abundant mRNA modification, N6-methyladenosine (m6A), is crucial for mammalian stem cell differentiation and tissue development in almost all systems tested so far. Dedicated proteins, many of which are essential in mammals, have evolved to install, recognize, and remove m6A marks (writers, readers, and erasers, respectively). Dysregulation of m6A methylation has been connected to a variety of human diseases and disorders. Functional roles have also been proposed for other internal modifications present in mammalian mRNA, including, but not limited to: pseudouridine (Ψ), 5-methylcytosine (m5C), 2’-O-methylation (Nm), N1-methyladenosine (m1A), N7- methylguanosine (m7G), and N3-methylcytosine (m3C). Our most recent research has uncovered modifications on chromosome-associated regulatory RNAs (carRNAs), such as promoter-associated RNA (paRNA), enhancer RNA (eRNA), and repeat RNAs, as well as frequent modifications in introns of pre-mRNA. The carRNA modifications have been shown to regulate chromatin state and transcription, and intron modifications may affect pre-mRNA processing. Despite rapid advances in the discovery and functional characterization of various RNA modifications and their effector proteins, a significant bottleneck limits the entire field of epitranscriptomics research: a dearth of quantitative sequencing methods that can comprehensively map most RNA modifications at base resolution with exact modification fraction information. The availability of such methods is critical for assessing the importance of these modifications in different regions of mRNA, examining the effects of dynamic changes in modification fraction, assigning modifications to different writers and analyzing their functional relevance, identifying target transcripts and sites of demethylation and analyzing their functional relevance, discovering new effectors for RNA modifications by overlapping with known RBP-binding sites or genomics features, and evaluating the physiological consequences of RNA modifications in biological processes. We have established both nucleic acid chemistry and directed protein evolution platforms to invent new technologies that transform RNA modifications to be read out as mutations or deletions that are universally compatible with extant sequencing platforms. Computational pipelines and RNA modification databases will be built to support the new method development and epitranscriptome research in the broad community. These new technologies will be optimized to work on low-input samples, particularly neuronal and clinical samples. We will focus on integrating new methods into robust protocols to map multiple RNA modifications in single experiments. Our proposed research will deliver high-throughput, high-resolution, and high-sensitivity methods that simultaneously map multiple RNA modifications in all biological areas.

项目总结/摘要 哺乳动物信使RNA（mRNA）上的化学修饰最近已被证明在哺乳动物的免疫反应中起关键作用， 在mRNA代谢和翻译中的多种调节作用。例如，最丰富的mRNA N6-甲基腺苷（m6 A）修饰对哺乳动物干细胞分化和组织形成至关重要。 到目前为止，几乎所有测试系统都在开发中。专门的蛋白质，其中许多是哺乳动物所必需的， 已经发展到安装、识别和删除m6 A标记（分别为写入器、读取器和橡皮擦）。 m6 A甲基化的失调与多种人类疾病和病症有关。功能 还提出了哺乳动物mRNA中存在的其他内部修饰的作用，包括但不限于 仅限于：假尿苷（pseudouridine，m5 C）、5-甲基胞嘧啶（5-methylcytosine，m5 C）、2 '-O-甲基化（2'-O-methylation，Nm）、N1-甲基腺苷（N1-methyladenosine，m1A）、N7- 甲基鸟苷（m7 G）和N3-甲基胞嘧啶（m3 C）。我们最近的研究发现 在染色体相关的调节RNA（carRNA）上，例如启动子相关RNA（paRNA）、增强子相关RNA（ERNA）、启动子相关RNA（ERNA）和增强子相关RNA（ERNA）上， RNA（eRNA）和重复RNA，以及前mRNA内含子中的频繁修饰。CarRNA 修饰已被证明可以调节染色质状态和转录，内含子修饰可能会影响 前mRNA加工。尽管各种RNA的发现和功能表征进展迅速， 修饰及其效应蛋白，一个重要的瓶颈限制了整个领域的epitranscriptomics 研究：缺乏可以全面绘制大多数RNA修饰的定量测序方法 在基础分辨率与准确的修改分数的信息。这些方法的可用性对于以下方面至关重要： 评估这些修饰在mRNA不同区域的重要性，检查动态修饰的影响， 修改分数的变化，将修改分配给不同的编写者并分析其功能 相关性，鉴定靶转录物和去甲基化位点并分析其功能相关性， 通过与已知的RBP结合位点或基因组重叠来发现新的RNA修饰效应子 特征，并评估生物过程中RNA修饰的生理后果。我们有 建立了核酸化学和定向蛋白质进化平台，以发明新技术， 将RNA修饰转化为突变或缺失，这些突变或缺失与现存的 测序平台。将建立计算管道和RNA修饰数据库，以支持新的 方法开发和epitranscriptome研究在广泛的社会。这些新技术将 优化以处理低输入样本，特别是神经元和临床样本。我们将专注于整合 新的方法转化为强大的协议，在单一实验中映射多种RNA修饰。我们提出的 研究将提供高通量、高分辨率和高灵敏度的方法， 在所有生物学领域都有多种RNA修饰。