Base-resolution mapping and site-specific epitranscriptomic studies in the brain

项目总结 神经发育和构成学习和记忆基础的突触可塑性事件 严重依赖严格控制的基因表达程序和快速、精细的翻译 信使RNA(信使核糖核酸)转录本。信使核糖核酸的降解、稳定和翻译 近年来，人们发现它受腺苷甲基化的调节，它改变了两种转录 结构和招募的RNA结合蛋白，通知这些活动。近期 甲基转移酶(“作者”)、去甲基酶的鉴定和实验 (“擦除器”)和特定的甲基腺苷结合蛋白(“阅读器”)已经确定 这些表位转录转录的mRNA调控过程既是动态的，也是严格调控的。 虽然这些修饰中研究最充分的是N6-甲基腺苷(M6A)，但N1- 甲基腺苷(M1a)最近也成为一种流行的表位转录标记。 目前用于探索这些修饰的方法需要大量的样本，并且 固有的低分辨率。这些限制使他们无法绘制和量化 在特定脑区或临床生物标本中的表位转录组。在这里，我们描述一下 创新技术的初步发展，以精确测序和探测 特定M6A和M1A修改的功能。我们建议利用这些基础在 服务于以下特定目标：1)进化并建立高分辨率、无抗体 用于脑分析的M6A和M1A映射平台，2)设计和验证分子工具包，以 在体内操纵转录特异性的m6A和M1a修饰，以及3)编目m6A和M1a 大脑在发育、神经元种群、活动状态和 突触，并决定它们与学习和记忆有关的功能。我们的发现将 阐明表位转录景观和特定的mRNA转录本是如何在离散的 神经元群体调节基因表达，以告知复杂的神经元过程，如 发育、学习和记忆及其干扰如何导致大脑异常 功能，如学习障碍。重要的是，我们的翻译、功能验证 将向研究界提供的新工具提供了强大的 为它们的使用奠定了基础，以具体询问mRNA修改是如何被干扰的 其他病理性的背景。

PROJECT SUMMARY Both neurodevelopment and the synaptic plasticity events that underlie learning and memory rely heavily on tightly regulated gene expression programs and rapid, finely-tuned translation of messenger RNA (mRNA) transcripts. The degradation, stability, and translation of mRNA has, in recent years, been found to be regulated by adenosine methylation, which alters both transcript structure and the recruitment of RNA-binding proteins that inform these activities. Recent identification of and experimentation with the methyltransferases (“writers”), demethylases (“erasers”), and specific methyladenosine binding proteins (“readers”) have established that these epitranscriptomic mRNA regulatory processes are both dynamic and tightly regulated. Although the most well-studied of these modifications is N6-methyladenosine (m6A), N1- methyladenosine (m1A) has also recently emerged as a prevalent epitranscriptomic mark. Current methods used to explore these modifications require large sample sizes and are inherently low-resolution. These limitations preclude them from mapping and quantifying the epitranscriptome in specific brain regions, or in clinical biospecimens. Here, we describe preliminary development of innovative technologies to precisely sequence and probe the function of specific m6A and m1A modifications. We propose to leverage these foundations in the service of the following specific aims: 1) Evolve and establish high-resolution, antibody-free m6A and m1A mapping platforms for brain analysis, 2) Design and validate a molecular toolkit to manipulate transcript-specific m6A and m1A modifications in vivo, and 3) Catalog m6A and m1A modifications in the brain across development, neuron populations, activity state, and in synapses, and determine their function in relation to learning and memory. Our findings will illuminate how the epitranscriptomic landscape and specific mRNA transcripts in discrete neuronal populations regulates gene expression to inform complex neuronal processes, such as development, learning and memory, and how perturbations thereof result in abnormal brain function such as learning impairment. Importantly, this translational, functional validation of our new tools, which will be made available to the research community, provides a strong foundation for their usage to specifically interrogate how mRNA modifications are perturbed in other pathological contexts.