Visible Light Activates Coumarin-Caged mRNA for Cytoplasmic Cap Methylation in Cells

Protein synthesis is important and regulated by various mechanisms in the cell. Translation initiation in eukaryotes starts at the 5' cap and is the most complex of the three phases of mRNA translation. It requires methylation of the N7 position of the terminal guanosine (m⁷ G). The canonical capping occurs in the nucleus, however, cytoplasmic recapping has been discovered. It functions in switching mRNAs between translating and non-translating states, but the individual steps are difficult to dissect. We targeted cytoplasmic cap methylation as the ultimate step of cytoplasmic recapping. We present an N7G photocaged 5' cap that can be activated for cytoplasmic methylation by visible light. We report chemical and chemo-enzymatic synthesis of this 5' cap with 7-(diethylamino)-4-methyl-coumarin (DEACM) at the N7G and validate that it is not bound by translation initiation factor 4E (eIF4E). We demonstrate incorporation into mRNA, the release of unmethylated cap analog and enzymatic remethylation to functional cap 0 after irradiation at 450 nm. In cells, irradiation triggers translation of mRNAs with the N7G photocaged 5' cap via cytoplasmic cap methylation.