The Regulation of mRNA Methylation by Glycogen Synthase Kinase-3

 描述(申请人提供)：Christopher J.Phiel，Ph.D.在40年前就发现了信使核糖核酸中存在甲基化的腺苷(称为m6A)，但m6A的功能意义仍然难以捉摸。最近的突破已经确定了建立和消除m6A标记的分子介体。此外，m6A在mRNA上的存在被发现是一个降解mRNA的信号。当胚胎干细胞(ESCs)中的m6A水平通过操纵m6A过程中涉及的因素而降低时，ESCs就不能正确分化，并保持多能状态。有趣的是，在胚胎干细胞中发现的无法分化的表型与基因GSK-3α和GSK-3β基因缺失的表型相同。葛兰素史克-3α和葛兰素史克-3β是磷酸化多种蛋白质的关键激酶。根据METTL3和GSK-3α/GSK-3β双基因敲除ESCs表型的相似性，我们推测M6A基因的修饰过程受α/GSK-3β活性的调控。具体地说，我们假设GSK-3α/GSK-3β正常情况下使m6A去甲基酶FTO磷酸化，靶向泛素介导的蛋白质降解；在没有GSK-3α/GSK-3β的情况下，FTO水平升高导致m6A水平降低，从而导致ESCs持续的多能性状态。为了验证这一假说，我们设计了以下特定目标：特定目标1.确定GSK-3活性调节ESCs FTO的确切机制。特定目的2.确定在GSK-3 DKO ESCs中减少m6A修饰的特定mRNAs。具体目的3.研究依赖GSK-3的信号通路对m6A水平的影响。这一发现表明，m6A修饰的mRNA是由一个多方面的信号转导调节因子控制的，这代表了对这一基本过程是如何调控的第一个机械性洞察，并将对理解不同的依赖于GSK-3的信号通路如何调节胚胎干细胞的多能性和分化之间的平衡具有深远的影响。

 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.