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

项目摘要 神经发育和作为学习和记忆基础的突触可塑性事件 严重依赖于严格调控的基因表达程序和快速，微调的翻译， 信使RNA（mRNA）转录物。mRNA的降解、稳定性和翻译， 近年来，发现腺苷甲基化可以调节，腺苷甲基化可以改变两种转录本， 结构和RNA结合蛋白的募集，告知这些活动。最近 甲基转移酶（"writers"）、脱甲基酶 （"擦除器"）和特异性甲基腺苷结合蛋白（"读取器"）已经确定， 这些表转录组mRNA调节过程是动态的和严格调节的。 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（m6A）是一种非结构化修饰。 甲基腺苷（m1A）最近也作为一种普遍的表观转录组学标记出现。 目前用于探索这些修改的方法需要大样本量， 固有的低分辨率。这些限制使他们无法绘制和量化 在特定脑区域或临床生物样本中的表转录组。在这里，我们描述 创新技术的初步开发，以精确测序和探测 特定m6A和m1A修饰的功能。我们建议利用这些基础， 服务的具体目标如下：1）发展和建立高分辨率，无抗体 用于大脑分析的m6A和m1A映射平台，2）设计和验证分子工具包， 在体内操纵转录物特异性m6A和m1A修饰，以及3）目录m6A和m1A 大脑在发育、神经元群体、活动状态和 突触，并决定其功能与学习和记忆。我们的发现将 阐明了如何epitranscriptomic景观和特定的mRNA转录在离散 神经元群体调节基因表达以告知复杂的神经元过程，例如 发展，学习和记忆，以及它们的扰动如何导致异常的大脑 功能，如学习障碍。重要的是，这种翻译，功能验证我们的 新的工具，这将提供给研究界，提供了一个强大的 他们的使用的基础，具体询问mRNA的修饰是如何扰动， 其他病理学背景。

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.