Glycogen synthase kinase-3 (Gsk-3) activity regulates multiple signal transduction pathways, and is also a key component of the network responsible for maintaining stem cell pluripotency. Genetic deletion of Gsk-3α and Gsk-3β or inhibition of Gsk-3 activity via small molecules promotes stem cell pluripotency, yet the mechanism underlying the role for Gsk-3 in this process remains ambiguous. Another cellular process that has been shown to affect stem cell pluripotency is mRNA methylation (m 6 A). Here, we describe an intersection between these components -the regulation of m 6 A by Gsk-3. We find that protein levels for the RNA demethylase, FTO (fat mass and obesity-associated protein), are elevated in Gsk-3α;Gsk-3β-deficient mouse embryonic stem cells (ESCs). FTO is normally phosphorylated by Gsk-3, and mass spectrometry identified the sites on FTO that are phosphorylated in a Gsk-3-dependent fashion. Gsk-3 phosphorylation of FTO leads to polyubiquitination, but in Gsk-3 knockout ESCs, that process is impaired, resulting in elevated levels of FTO protein. As a consequence of altered FTO protein levels, mRNAs in Gsk-3 knockout ESCs have 50% less m 6 A than wild-type ESCs, and m 6 A-seq analysis reveals the specific mRNAs that have reduced m 6 A modifications. Taken together, we provide the first evidence for how m 6 A demethylation is regulated in mammalian cells, and sheds light onto a possible novel mechanism by which Gsk-3 activity regulates stem cell pluripotency.Glycogen synthase kinase-3 (Gsk-3) activity is an important regulator of numerous signal transduction pathways (1). Gsk-3 activity is the sum of two largely redundant proteins, Gsk-3α and Gsk-3β, and in general, Gsk-3 is a negative regulator of cellular signaling (2). Rare among kinases, Gsk-3 is active at a basal state, while pathway activation from upstream signaling cascades results in the inhibition of Gsk-3 activity (2). Gsk-3α and Gsk-3β together regulate signal transduction pathways such as Wnt, protein kinase A (PKA), Hedgehog, transforming growth factor-β (TGF-β), nuclear factor of activated T-cells (NF-AT) and phosphatidylinositol 3-kinase (PI3K)-dependent insulin signaling in a variety of biological settings (3)(4)(5).Gsk-3 activity can be inhibited through the use of small molecule inhibitors, such as SB- (6)(7)(8), and the clinically relevant mood-stabilizer lithium (9,10); however, a drawback to the use of small molecules to study Gsk-3 function is the potential for off-target effects (11). Cells in which Gsk-3α and Gsk-3β have been genetically deleted allows for a more confident assessment of Gsk-3-specific functions. Therefore, we utilize mouse embryonic stem cells (ESCs) deficient in both Gsk-3α and Gsk-3β (Gsk-3α -/-; Gsk-3β -/-), i.e., Gsk-3 double knockout (DKO), to assess Gsk-3-specific functions (12,13). One prominent phenotype of Gsk-3 DKO ESCs is their persistent pluripotency, assessed by their inability to differentiate into all three germs layers in a teratoma assay, as well as by analysis of global gene expression ...
The pluripotency of embryonic stem cells (ESCs) is actively promoted by a diverse set of factors, including leukemia inhibitory factor (LIF), glycogen synthase kinase-3 (Gsk-3) and mitogen-activated protein kinase kinase (MEK) inhibitors, ascorbic acid, and alpha-ketoglutarate. Strikingly, several of these factors intersect with the post-transcriptional methylation of RNA (m6A), which has also been shown to play a role in ESC pluripotency. Therefore, we explored the possibility that these factors converge on this biochemical pathway to promote the retention of ESC pluripotency. Mouse ESCs were treated with various combinations of small molecules, and the relative levels of m6A RNA were measured, as well as the expression of genes marking naive and primed ESCs. The most surprising result was the discovery that replacing glucose with high levels of fructose pushed ESCs to a more naive state and reduced m6A RNA abundance. Our results suggest a correlation between molecules previously shown to promote the retention of ESC pluripotency and m6A RNA levels, strengthening a molecular connection between reduced m6A RNA and the pluripotent state, and provides a foundation for future mechanistic studies on the role of m6A and ESC pluripotency.
Methylation of mRNA on adenosine bases (referred to as m6A) is the most common internal modification of mRNA in eukaryotic cells. Recent work has revealed a detailed view of the biological significance of m6A-modified mRNA, with a role in mRNA splicing, control of mRNA stability, and mRNA translation efficiency. Importantly, m6A is a reversible modification, and the primary enzymes responsible for methylating (Mettl3/Mettl14) and demethylating RNA (FTO/Alkbh5) have been identified. Given this reversibility, we are interested in understanding how m6A addition/removal is regulated. Recently, we identified glycogen synthase kinase-3 (Gsk-3) activity as a mediator of m6A regulation via controlling the levels of the FTO demethylase in mouse embryonic stem cells (ESCs), with Gsk-3 inhibitors and Gsk-3 knockout both leading to increased FTO protein and decreased m6A mRNA levels. To our knowledge, this remains one of the only mechanisms identified for the regulation of m6A modifications in ESCs. Several small molecules that have been shown to promote the retention of pluripotency of ESCs, and interestingly, many have connections to the regulation of FTO and m6A. Here we show that the combination of Vitamin C and transferrin potently reduces levels of m6A and promotes retention of pluripotency in mouse ESCs. Combining Vitamin C and transferrin should prove to be valuable in growing and maintaining pluripotent mouse ESCs.
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