Capture and identification of the eIF4E:mRNA interactome in human cells

Here we develop a novel methodology, capCLIP, to capture and identify mRNA interactions with the major cellular cap-binding protein eIF4E. HeLa cells in which a 3X-flag epitope tag has been introduced on to the N-terminal end of endogenous eIF4E protein were treated with either 100 nM rapamycin (or DMSO carrier along) for 2 hours, and then subject to 350 mJ/cm2 of UV irradiation to crosslink eIF4E to the m7G cap of mRNA. Biological replicates have been done for each treatment condition. Hela cells in which a 3X-flag epitope tag has been introduced on to the N-terminal end of endogenous eIF4E protein were serum-starved overnight then pre-treated for 1 hour with either 100 nM eFT-508 or with vehicle DMSO. Cells were then stimulated for 30 minutes with 500 nM PMA to activate MAP kinase signalling and thus MNK activity, and then subject to 350 mJ/cm2 of UV irradiation to crosslink eIF4E to the m7G cap of mRNA. Biological replicates have been done for each treatment condition.