Mechanistic differences in eukaryotic initiation factor requirements for eIF4GI-driven cap-independent translation of structured mRNAs

Abstract

Protein translation is globally downregulated under stress conditions. Many proteins that are synthesized under stress conditions use a cap-independent translation initiation pathway. A subset of cellular mRNAs that encode for these proteins contain stable secondary structures within their 5’ untranslated region (5’UTR), and initiate cap-independent translation using elements called Cap-Independent Translation Enhancers (CITEs) or Internal Ribosome Entry Sites (IRESs) within their 5’UTRs. The interaction among initiation factors such as eIF4E, eIF4A and eIF4GI, especially in regulating the eIF4F complex during non-canonical translation initiation of different 5’UTR mRNAs, is poorly understood. Here, equilibrium-binding assays, circular dichroism studies and in vitro translation assays were employed to elucidate the recruitment of these initiation factors to the highly structured 5’UTRs of fibroblast-growth factor 9 (FGF-9) and hypoxia inducible factor 1 subunit alpha (HIF-1α) encoding mRNAs. We showed that eIF4A and eIF4E enhanced eIF4GI’s binding affinity to the uncapped 5’UTR of HIF-1α mRNA, inducing conformational changes in the protein/RNA complex. In contrast, these factors have no effect on the binding of eIF4GI to the 5’UTR of FGF-9 mRNA. Recently, Izidoro, M. S. et al. reported that the interaction of 42nt unstructured RNA to human eIF4F complex is dominated by eIF4E and ATP-bound state of eIF4A. Here we show that structured 5’UTR mRNA binding mitigates this requirement. Based on these observations, we describe two possible cap-independent translation mechanisms for FGF-9 and HIF-1α encoding mRNAs employed by cells to mitigate cellular stress conditions.