RNA-tethering assay and eIF4G:eIF4A obligate dimer design uncovers multiple eIF4F functional complexes

Eukaryotic cellular mRNAs possess a 5′ cap structure (m7GpppN) which plays a critical role in translation initiation mediated by eukaryotic initiation factor (eIF) 4F. The heterotrimeric eIF4F complex possesses several activities imparted by its subunits that include cap recognition (by eIF4E), RNA unwinding (eIF4A), and factor/ribosome recruitment (eIF4G). Mammalian cells have paralogs of all three eIF4F subunits and it remains an open question as to whether these all can participate in the process of ribosome recruitment. To query the activities of the eIF4F subunits in translation initiation, we adopted an RNA-tethering assay in which select subunits are recruited to a specific address on a reporter mRNA template. We find that all eIF4F subunits can participate in the initiation process. Based on eIF4G:eIF4A structural information, we also designed obligate dimer pairs to probe the activity of all combinations of eIF4G and eIF4A paralogs. We demonstrate that both eIF4GI and eIF4GII can associate with either eIF4A1 or eIF4A2 to recruit ribosomes to mRNA templates. In combination with eIF4E and eIF4E3, our results indicate the presence of up to eight eIF4F complexes that can operate in translation initiation.