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-3 and Gsk-3 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-3;Gsk-3-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 m(6)A than WT 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 identify a putative novel mechanism by which GSK-3 activity regulates stem cell pluripotency.