Quantitative imaging of epitranscriptomic regulation mediated by RNA modification

项目摘要 RNA修饰是调控基因表达的新的表观转录标记，对 它与人类疾病有关，如神经性疾病和癌症。N6-甲基腺苷 (M6A)是信使RNA(MRNA)和长非编码RNA(LncRNA)中最常见的修饰 高等真核生物。M6A在目标RNAs上动态安装和删除，并可由 各种m6A阅读器蛋白(包含YTH结构域的蛋白质，或YTH蛋白)来调节靶RNA 通过不同的机制，或改变局部RNA的结构来微调RNA-蛋白质的相互作用。多么 一种单一类型的修饰是否允许以不同的方式调节一个RNA靶标？什么是 有助于选择特定RNA的特定调控机制的信号？我们假设 由于存在多个甲基化位点，单个RNA可以存在于多个甲基化状态 以及每个位点的不完全甲基化。甲基化状态起到“表位转录密码”的作用，并且可以 决定核糖核酸的命运。 单个RNA的甲基化状态以及甲基化状态与不同物种之间的机制联系 M6A介导的调节通路只能通过实验方法进行剖析，这种方法既具有特异性，又具有 确定了单个RNA的甲基化位点和敏感性。然而，这种实验性的方法目前正在 无法获得，这阻碍了我们目前理解RNA修饰的作用的努力。来解决这个问题 问题是，在目标1中，我们将首先开发一套具有单RNA敏感性和单RNA敏感性的成像平台。 甲基化位点特异性。利用这些独特的技术，我们将重点研究lncrna和mrna。 系统。在目标2中，我们将研究lncRNA MALAT1的甲基化状态是否对其 多价结合能力，它的亚细胞定位，以及它对活跃转录的基因位点的招募 促进拼接。在目标3中，我们将探索mRNA中的甲基化状态如何影响选择 翻译或降解水平的调控途径，以及m6A介导的调控的强度。 我们提出的体内单个RNA水平的RNA修饰实验将提供有价值的机制 洞察m6A在lncRNA和mRNA系统中的调节。在这样的先驱和新颖的成像中 平台通常也可以用来研究其他的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.