Vitamin C and Transferrin Reduce RNA Methylation in Mouse Embryonic Stem Cells.

Methylation of mRNA on adenosine bases (referred to as m 6 A) is the most common internal modification of mRNA in eukaryotic cells. Recent work has revealed a detailed view of the biological significance of m 6 A-modified mRNA, with a role in mRNA splicing, control of mRNA stability, and mRNA translation efficiency. Importantly, m 6 A 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 m 6 A addition/removal is regulated. Recently, we identified glycogen synthase kinase-3 (Gsk-3) activity as a mediator of m 6 A 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 m 6 A mRNA levels. To our knowledge, this remains one of the only mechanisms identified for the regulation of m 6 A 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 m 6 A. Here we show that the combination of Vitamin C and transferrin potently reduces levels of m 6 A 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.

Mouse Embryonic Stem Cell Pluripotency Factors Regulate RNA Methylation.

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 α-ketoglutarate. Strikingly, several of these factors intersect with the post-transcriptional methylation of RNA (m 6 A), 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 m 6 A RNA were measured, as well as the expression of genes marking naïve and primed ESCs. The most surprising result was the discovery that replacing glucose with high levels of fructose pushed ESCs to a more naïve state and reduced m 6 A RNA abundance. Our results suggest a correlation between molecules previously shown to promote the retention of ESC pluripotency and m 6 A RNA levels, strengthening a molecular connection between reduced m 6 A RNA and the pluripotent state, and provides a foundation for future mechanistic studies on the role of m 6 A and ESC pluripotency.

Glycogen synthase kinase-3 (GSK-3) activity regulates mRNA methylation in mouse embryonic stem cells.

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-3ß 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 (m6A). Here, we describe an intersection between these components, the regulation of m6A 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-3ß-deficient mouse embryonic stem cells (ESCs). FTO is normally phosphorylated by GSK-3, and MS 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 m6A than WT ESCs, and m6A-Seq analysis reveals the specific mRNAs that have reduced m6A modifications. Taken together, we provide the first evidence for how m6A demethylation is regulated in mammalian cells and identify a putative novel mechanism by which GSK-3 activity regulates stem cell pluripotency.