Center for dynamic RNA epitranscriptomes - Renewal

项目摘要/摘要 最近，对哺乳动物信使RNA(MRNA)的化学修饰被证明发挥着关键和 在信使核糖核酸代谢和翻译中的不同调节作用。例如，最丰富的mRNA 修饰N6-甲基腺苷(M6A)对哺乳动物干细胞分化和组织至关重要 到目前为止，几乎所有经过测试的系统都在开发。专门的蛋白质，其中许多是哺乳动物所必需的， 已经进化到安装、识别和移除M6A标记(分别是写入器、读取器和橡皮擦)。 M6A甲基化的失调与人类的各种疾病和障碍有关。功能性 哺乳动物中存在的其他内部修饰也被提出了作用，包括，但不是 仅限于：假尿苷(Ψ)、5-甲基胞嘧啶(M5C)、2‘-O-甲基化(Nm)、N1-甲基腺苷(M1a)、N7- 甲基鸟苷(M7G)和N3-甲基胞嘧啶(M3C)。我们最新的研究发现了 关于染色体相关调控RNA(CarRNAs)，如启动子相关RNA(PARNA)、增强子 RNA(Erna)和重复RNA，以及Pre-mRNA内含子的频繁修改。核糖核糖核酸 已证明修饰可以调节染色质状态和转录，内含子修饰可能会影响 前信使核糖核酸加工。尽管在各种RNA的发现和功能表征方面取得了快速进展 修饰及其效应蛋白，一个重要的瓶颈限制了表位转录组学的整个领域 研究：缺乏能够全面定位大多数RNA修饰的定量测序方法 在基本分辨率下，具有精确的修饰分数信息。这些方法的可用性对于 评估这些修饰在不同的信使核糖核酸区域的重要性，检查动态的影响 修改比例的变化，将修改分配给不同的编写者并分析其功能 相关性，识别目标转录本和去甲基化位点并分析它们的功能相关性， 通过与已知的RBP结合位点或基因组学重叠发现RNA修饰的新效应物 特征，并评估生物过程中RNA修饰的生理后果。我们有 建立核酸化学和定向蛋白质进化平台，以发明新技术 将待读出的RNA修改转换为与现有的普遍兼容的突变或缺失 测序平台。将建立计算管道和RNA修改数据库，以支持新的 在广泛的社区中开展方法开发和表型转录组研究。这些新技术将成为 针对低输入样本进行了优化，特别是神经元和临床样本。我们将专注于整合 将新方法引入稳健的协议中，以在单个实验中映射多个RNA修改。我们的建议 研究将提供高通量、高分辨率和高灵敏度的方法，同时绘制 所有生物领域的多重RNA修饰。

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.