Quantitative imaging of epitranscriptomic regulation mediated by RNA modification

项目摘要 RNA修饰是调节基因表达的新兴表观转录组学标记，并且对于调节基因表达至关重要。 发展和人类疾病的联系，如神经元疾病和癌症。N6-甲基腺苷 (m6A)是信使RNA（mRNA）和长链非编码RNA（lncRNA）中最普遍的修饰， 高等真核生物m6 A在靶RNA上动态地安装和移除，并且可以被 调节靶RNA的各种m6 A阅读器蛋白（含有YTH结构域的蛋白，或YTH蛋白 通过不同的机制，或改变局部RNA结构来微调RNA-蛋白质相互作用。如何 一种单一的修饰是否允许一种RNA靶以不同的方式被调节？有哪些 信号有助于选择一个特定的调节机制，为一个特定的RNA？我们假设 由于存在多个甲基化位点，单个RNA可以以多种“甲基化状态”存在， 和不完全甲基化。甲基化状态充当“表位转录组密码”， 决定RNA的命运。 单个RNA的甲基化状态以及甲基化状态与各种RNA之间的机制联系， m6 A介导的调节途径只能通过实验方法进行分析， 确定的甲基化位点和单个RNA的敏感性。然而，这种实验方法目前 这阻碍了我们目前理解RNA修饰作用的努力。解决这个 问题，在目标1中，我们将首先开发一套具有单RNA灵敏度和单 甲基化位点特异性。利用这种独特的技术，我们将重点研究lncRNA和mRNA 系统.在目标2中，我们将研究lncRNA，MALAT 1的甲基化状态是否影响其表达。 多价结合能力，其亚细胞定位，以及其对活跃转录基因位点的募集， 促进剪接。在目标3中，我们将探索mRNA中的甲基化状态如何影响对基因的选择。 翻译或降解水平的调控途径，以及m6 A介导调控的强度。 我们提出的在体内单RNA水平上进行RNA修饰的实验将提供有价值的机制， 对lncRNA和mRNA系统中m6 A介导调节的见解。 这种开创性和新颖的成像 这些平台通常也可以适用于研究其他RNA修饰。

Project Summary RNA modifications are emerging epitranscriptomic markers in regulating gene expression, and are crucial for development and linked to human diseases, such as neuronal disorders and cancers. N6-methyladenosine (m6A) is the most prevalent modification in the messenger RNA (mRNA) and long noncoding RNA (lncRNA) in higher eukaryotes. m6A are dynamically installed and removed on the target RNAs, and can be recognized by various m6A reader proteins (proteins containing YTH domains, or YTH proteins) to regulate the target RNAs through diverse mechanisms, or alter the local RNA structure to fine tune the RNA-protein interactions. How does one single type of modification allow one RNA target to be regulated in diverse ways? What are the signals contributing to the choice of a specific regulatory mechanism for a particular RNA? We hypothesize that a single RNA can exist in multiple “methylation states” due to the existence of multiple methylation sites and incomplete methylation at each site. The methylation state serves as “epitranscriptomic code” and may determine fate of the RNA. The methylation states of a single RNA and the mechanistic linkage between methylation states and various m6A-mediated regulation pathways can only be dissected by experimental approaches with both specificity to a defined methylation site and sensitivity of single RNA. However, such experimental approaches are currently unavailable, which hampers our current efforts to understand the role of RNA modifications. To tackle this problem, in Aim 1, we will first develop a suite of imaging platforms with single-RNA sensitivity and single- methylation site specificity. With the unique techniques, we will then focus on studying lncRNA and mRNA systems. In Aim 2, we will investigate whether methylation state of an lncRNA, MALAT1, has impact on its multivalent binding capacity, its subcellular localization, and its recruitment to actively transcribed gene loci to promote splicing. In Aim 3, we will explore how methylation state in an mRNA can influence choice of regulatory pathways at translation or degradation levels, and the strength of m6A-mediate regulation. Our proposed experiments on RNA modification at single-RNA level in vivo will provide valuable mechanistic insight of m6A-mediated regulation in both lncRNA and mRNA systems.  Such pioneer and novel imaging platforms can be also generally adapted to study other RNA modifications.