Molecular and Cellular Biology (360G-Wellcome-097462_Z_11_Z)

The methyl cap is a structure added to mRNA, which mediates processing and translation initiation. Synthesis of the methyl cap is catalysed by two enzymes: CE and RNMT. We are investigating the pathways that regulate these enzymes and methyl cap synthesis by characterising CE and RNMT interacting proteins and post-translational modifications. My host lab discovered that synthesis of the methyl cap is regulated by c-Myc oncogene in mammary epithelial cells. Using mass spectrometry, I identified phosphorylated residues on the cap methyltransferase, some of which are c-Myc-dependent in mammary epithelial cells. In order to determine whether these phosphorylation events are functionally significant, mammary epithelial cell lines will be created in which the modified amino acids are mutated to alanine, to prevent their phosphorylation, and the effect on RNMT expression, activity and localisation will be assessed. If these assays suggest that the phosphorylation events are functionally significant, we will raise antibodies against the phosphorylated residues, and use a panel of inhibitors to dissect the kinase and signalling pathway responsible. Simultaneously, methyl cap formation will be studied for the first time in mouse embryonic stem (ES) cells, investigating the role of cap methylation in self-renewal and cell differentiation.