The Regulation of mRNA Methylation by Glycogen Synthase Kinase-3

Christopher J. Phiel博士 胚胎干细胞（ESC）是哺乳动物细胞中的独特细胞，由于它们能够分化成任何细胞。 细胞类型，一个称为多能性的过程。为了保持多能性，胚胎干细胞还必须抵抗外部信号 促进细胞分化。考虑到胚胎干细胞对胚胎发育的重要性， 未来再生疗法的发展，充分了解胚胎干细胞如何保留的分子基础 他们的多能状态引起了人们的极大兴趣。几年前，研究表明，抑制丝氨酸/苏氨酸蛋白的表达， 糖原合成酶激酶-3（Gsk-3）促进ESC多能性，但Gsk-3的确切作用 在这种情况下，抑制作用很难阐明。 另一个已被证明可以调节ESC多能性的细胞过程是mRNA甲基化减少 （简称m6 a）。在我们以前授予的赠款中，我们建立了一种新的机制， m6 A mRNA-GSK-3直接磷酸化RNA去甲基化酶FTO，靶向FTO用于随后的脱甲基化。 通过蛋白酶体的泛素化和降解。在缺乏Gsk-3α和Gsk-3β（Gsk-3双链）的情况下， 在小鼠ESC中，FTO磷酸化和泛素化受损，导致FTO磷酸化和泛素化增加。 FTO水平和伴随的m6 A mRNA量减少50%。综合起来，我们的数据直接 将ESC多能性的两个看似无关的方面联系起来;然而，关于ESC多能性调控的许多细节， mRNA甲基化仍有待研究。在这里，我们提出调查三个突出的问题。 1.）目前尚不清楚GSK-3上游信号通路的激活是否会导致随后的变化 mRNA甲基化。我们打算研究Wnt或磷脂酰肌醇-3激酶（PI 3 K）信号转导， 两者都已知调节ESC多能性，并且都已知抑制GSK-3活性，也导致 mRNA甲基化。此外，最近显示，通过Smad 2/3的信号传导下调Mettl 3，其表达下调。 促进m6 A的RNA甲基转移酶。我们将测试是否通过Smad 2/3激活减少Mettl 3， 与通过Gsk-3缺失或抑制增加的FTO结合，更有效地推动ESC， 多能性。2.）出乎意料的是，最近的研究表明，克雷布斯循环中间体α- 酮戊二酸，在GSK-3和促分裂原活化蛋白激酶激酶（MEK）抑制剂存在下， 增强了胚胎干细胞的多能性。有趣的是，α-酮戊二酸也需要作为FTO的辅助因子。 因此，我们打算检查α-酮戊二酸是否通过减少mRNA来促进多能性， 甲基化3.）第三章据报道，FTO优先去甲基二甲基腺苷（m6 Am），但我们 尚未具体研究Gsk-3 DKO ESC中是否存在任何m6 Am变化。沿着着 科罗拉多大学医学院的一位同事，他一直在使用m6 A单个核苷酸， 分辨率交联和免疫沉淀（miCLIP），我们打算检查野生型（WT）中的m6 Am， GSK-3 DKO ESC。 该项目旨在让本科生有意义的参与。了解GSK-3 与mRNA甲基化和细胞代谢的交叉应该提供一个更完整的图片， 胚胎干细胞多能性的基本机制。

Christopher J. Phiel, Ph.D. Embryonic stem cells (ESCs) are unique among mammalian cells due to their ability to differentiate into any cell type, a process termed pluripotency. In order to remain pluripotent, ESCs must also resist external cues that promote cellular differentiation. Given the importance of ESCs to embryonic development, as well as for the development of future regenerative therapies, fully understanding the molecular basis of how ESCs retain their pluripotent state is of keen interest. Several years ago, it was shown that inhibition of the serine/threonine kinase, glycogen synthase kinase-3 (Gsk-3), promotes ESC pluripotency, yet the precise role for Gsk-3 inhibition in this context has been difficult to elucidate. Another cellular process that has been shown to regulate ESC pluripotency is reduced mRNA methylation (referred to as m6A). In our previously awarded grant, we established a novel mechanism for the regulation of m6A mRNA - Gsk-3 directly phosphorylates the RNA demethylase FTO, targeting FTO for subsequent ubiquitination and degradation via the proteasome. In the absence of Gsk-3α and Gsk-3β (Gsk-3 double knockout; DKO) in mouse ESCs, FTO phosphorylation and ubiquitination are impaired, leading to increased FTO levels and a concomitant 50% decrease in the amount of m6A mRNA. Taken together, our data directly link two seemingly unrelated aspects of ESC pluripotency; however, many details about the regulation of mRNA methylation remain to be investigated. Here, we propose to investigate three prominent questions. 1.) It is unknown whether the activation of signaling pathways upstream of Gsk-3 result in subsequent changes in mRNA methylation. We intend to investigate whether Wnt or phosphatidylinositol-3 kinase (PI3K) signaling, both known to regulate ESC pluripotency and both known to inhibit Gsk-3 activity, also leads to changes in mRNA methylation. In addition, it was recently shown that signaling via Smad2/3 down-regulates Mettl3, the RNA methyltransferase that promotes m6A. We will test whether a decrease in Mettl3 via Smad2/3 activation, in combination with increased FTO via Gsk-3 deletion or inhibition, more efficiently pushes ESCs to pluripotency. 2.) Unexpectedly, it was recently shown that elevation of the Krebs cycle intermediate α- ketoglutarate, in the presence of Gsk-3 and mitogen-activated protein kinase kinase (MEK) inhibitors, enhanced the pluripotency of ESCs. Interestingly, α-ketoglutarate is also required as a co-factor for FTO. Therefore, we intend to examine whether α-ketoglutarate promotes pluripotency via reducing mRNA methylation. 3.) It has been reported that FTO preferentially demethylates dimethyladenosine (m6Am), yet we have not specifically investigated whether there are any m6Am changes in Gsk-3 DKO ESCs. Along with a colleague at the University of Colorado School of Medicine who has been using m6A individual-nucleotide- resolution cross-linking and immunoprecipitation (miCLIP), we intend to examine m6Am in wild-type (WT) and Gsk-3 DKO ESCs. This project is designed to allow the meaningful participation of undergraduates. Understanding how Gsk-3 intersects with mRNA methylation and cellular metabolism should provide a more complete picture of the fundamental mechanisms underlying embryonic stem cell pluripotency.