eIF4A alleviates the translational repression mediated by classical secondary structures more than by G-quadruplexes

Increased activity of the mRNA helicase eIF4A drives cellular malignancy by reprogramming cellular translation, and eIF4A activity is the direct or indirect target of many emerging cancer therapeutics. The enriched presence of (GGC)4 motifs, which have the potential to fold into two-layered G-quadruplexes, within the 5′UTRs of eIF4A-dependent mRNAs suggests that eIF4A is required for the unwinding of these structures within these eIF4A-controlled mRNAs. However, the existence of folded G-quadruplexes within cells remains controversial, and G-quadruplex folding is in direct competition with classical Watson–Crick based secondary structures. Using a combination of reverse transcription stalling assays and 7-deazaguanine incorporation experiments we find that (GGC)4 motifs preferentially form classical secondary structures rather than G-quadruplexes in full-length mRNAs. Furthermore, using translation assays with the eIF4A inhibitor hippuristanol, both in vitro and in cells, we find that eIF4A activity alleviates translational repression of mRNAs with 5′UTR classical secondary structures significantly more than those with folded G-quadruplexes. This was particularly evident in experiments using a G-quadruplex stabilizing ligand, where shifting the structural equilibrium in favour of G-quadruplex formation diminishes eIF4A-dependency. This suggests that enrichment of (GGC)4 motifs in the 5′UTRs of eIF4A-dependent mRNAs is due to the formation of stable hairpin structures rather than G-quadruplexes.