The Regulation of mRNA Methylation by Glycogen Synthase Kinase-3

 描述（由申请人提供）：Christopher J. Phiel，Ph.D. mRNA中存在甲基化腺苷（简称m6 A）的现象早在40年前就已被发现，但m6 A的功能意义仍不清楚。最近的突破已经确定了建立和消除m6 A标记的分子介质。此外，发现mRNA上m6 A的存在是降解mRNA的信号。当通过操纵参与m6 A过程的因子而降低胚胎干细胞（ESC）中的m6 A水平时，ESC不能正确分化，并保持多能状态。有趣的是，无法分化与ESCs中发现的Gsk-3α和Gsk-3β基因缺失的表型相同。GSK-3α和GSK-3β是磷酸化多种蛋白质的关键激酶。基于Mettl 3和Gsk-3α/Gsk-3β双敲除（DKO）ESCs在表型上的相似性，我们推测mRNA m6 A的修饰过程受Gsk-3α/Gsk-3β活性的调控。具体来说，我们假设Gsk-3α/Gsk-3β通常磷酸化m6 A脱甲基酶FTO，靶向蛋白质进行泛素介导的降解;在Gsk-3α/Gsk-3β不存在的情况下，FTO水平升高，导致m6 A水平降低，导致ESC中持续的多能状态。以下具体目标旨在检验这一假设：具体目标1。确定GSK-3活性调节ESCs中FTO的确切机制。具体目标2。鉴定在Gsk-3 DKO ESC中具有减少的m6 A修饰的特定mRNA。具体目标3。研究哪些GSK-3依赖的信号通路影响m6 A水平。发现m6 A修饰的mRNA是由多方面的信号转导调节器控制，代表了第一个机制的洞察如何调节这一基本过程，并将有深刻的影响，了解不同的GSK-3依赖性信号通路如何调节胚胎干细胞多能性和分化之间的平衡。

 DESCRIPTION (provided by applicant): Christopher J. Phiel, Ph.D. The presence of methylated adenosines (referred to as m6A) in mRNA had been discovered 40 years ago, but the functional significance of m6A remained elusive. Recent breakthroughs have identified the molecular mediators that establish and remove the m6A mark. In addition, the presence of m6A on mRNA was found to be a signal to degrade mRNA. When m6A levels are reduced in embryonic stem cells (ESCs) by manipulation of the factors involved in the m6A process, the ESCs are unable to properly differentiate, and remain in a pluripotent state. Interestingly, the inability to differentiate is the same phenotype found in ESCs in which the genes Gsk-3α and Gsk-3β are genetically deleted. Gsk-3α and Gsk-3β are key kinases that phosphorylate a variety of proteins. Based on the similarity in phenotypes between Mettl3 and Gsk-3α/Gsk-3β double knockout (DKO) ESCs, we hypothesize that the process of mRNA m6A modification is regulated by Gsk-3α/Gsk-3β activity. Specifically, we hypothesize that Gsk-3α/Gsk-3β normally phosphorylates the m6A demethylase FTO, targeting the protein for ubiquitin-mediated degradation; in the absence of Gsk-3α/Gsk-3β, FTO levels are raised resulting in reduced m6A levels, leading to a persistent pluripotent state in ESCs. The following specific aims are designed to test this hypothesis: Specific Aim 1. Determine the precise mechanism by which Gsk-3 activity regulates FTO in ESCs. Specific Aim 2. Identify the specific mRNAs that have reduced m6A modifications in Gsk-3 DKO ESCs. Specific Aim 3. Investigate which Gsk-3-dependent signaling pathways affect m6A levels. The discovery that the m6A modification of mRNA is controlled by a multi-faceted regulator of signal transduction represents the first mechanistic insight into how this fundamental process is regulated, and would have profound consequences for understanding how different Gsk-3-dependent signaling pathways regulate the balance between pluripotency and differentiation in embryonic stem cells.