The interactome of flavin adenine dinucleotide (FAD) with RNA-binding proteins

We explored the interactome of flavin adenine dinucleotide (FAD) and the matrix of UV-immobilised FAD bound more than 3,000 proteins from SW-620 lysate. GO enrichment analysis showed that about 600 RNA-binding proteins were bound to FAD beads and, surprisingly, more than 40 proteins showed clear dose-dependent binding competition with nanomolar affinities indicating that their direct or indirect interaction with free FAD is both specific and strong. Among the few known consensus sequences for RNA-binding proteins, we selected the PUF motif 5'-UGUANAUA-3' to investigate whether FAD is binding the Pumilio homologs PUM1 and PUM2 in their RNA-binding pocket, as both these proteins showed dose-response behaviour in the FAD versus FAD-beads. Immobilisation of an oligomer containing the consensus sequence on NHS-activated Sepharose beads yielded PUM-beads that did enrich the Pumilio homologs. Oligomer vs FAD-beads and FAD vs PUM-beads together with the FAD vs FAD-beads dose-response competition indicated that the binding of FAD does not seem to influence the binding of the RNA. It is therefore most likely that FAD binding is allosteric for PUMs.