The Regulation of mRNA Methylation by Glycogen Synthase Kinase-3

克里斯托弗·J·菲尔博士。 胚胎干细胞(ESCs)在哺乳动物细胞中是独一无二的，因为它们能够分化为 细胞类型，这一过程称为多能性。为了保持多能性，胚胎干细胞还必须抵抗外界的暗示。 促进细胞分化的物质。鉴于胚胎干细胞对胚胎发育的重要性，以及对 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 他们的全能状态引起了人们的极大兴趣。几年前，研究表明，丝氨酸/苏氨酸的抑制 糖原合成酶激酶-3(GSK-3)促进胚胎干细胞的多能性，但其确切的作用 在这种情况下，抑制作用一直很难阐明。 另一个已被证明调节胚胎干细胞多能性的细胞过程是减少的mRNA甲基化 (称为M6A)。在我们之前授予的拨款中，我们建立了一个新的机制来监管 M6A mRNA-GSK-3直接磷酸化RNA去甲基酶FTO，靶向FTO 通过蛋白酶体泛素化和降解。在没有葛兰素史克-3α和葛兰素史克-3β(葛兰素史克-3双 在小鼠胚胎干细胞中，FTO磷酸化和泛素化受到损害，导致 FTO水平和伴随的m6A mRNA数量减少50%。总而言之，我们的数据直接 将ESC多能性的两个看似无关的方面联系在一起；然而，关于监管的许多细节 信使核糖核酸的甲基化还有待研究。在这里，我们建议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后续的变化 在信使核糖核酸甲基化。我们打算研究Wnt或磷脂酰肌醇-3激酶(PI3K)信号转导途径， 两者都已知调节ESC的多能性，都已知抑制GSK-3的活性，也导致 MRNA甲基化。此外，最近研究表明，通过Smad2/3发出的信号下调了METTL3，即 促进M6A的RNA甲基转移酶。我们将测试是否通过激活Smad2/3来减少METTL3， 与通过GSK-3缺失或抑制而增加的FTO相结合，更有效地推动ESCs 多能性。2.)出乎意料的是，最近的研究表明，克雷布斯旋回的高程介于α- 酮戊二酸，在GSK-3和丝裂原活化蛋白激酶(MEK)抑制剂存在的情况下， 增强胚胎干细胞的多能性。有趣的是，α-酮戊二酸也是FTO的辅助因素。 因此，我们打算研究α-酮戊二酸是否通过减少mrna来促进多能性。 甲基化。3.)据报道，FTO优先去甲基化二甲基腺苷(M6Am)，但我们 还没有具体调查GSK-3 DKO ESCs是否有任何m6AM变化。以及一个 他是科罗拉多大学医学院的一位同事，他一直在使用m6A个人核苷酸- 为了检测m6Am在野生型(WT)和野生型(WT)中的分布 GSK-3 DKO ESCs。 这个项目旨在让本科生有意义地参与其中。了解GSK-3如何 与mRNA甲基化和细胞新陈代谢的交叉应提供更完整的图景 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.