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Initiation of translation for the majority of eukaryotic mRNAs is mediated by a 50 cap structure to which the eukaryotic initiation factor 4E (eIF4E) binds. Inhibition of the activity of eIF4E by 4EBP-1 does not prevent the translation of a number of cellular capped mRNAs, indicative of the existence of previously unexplored mechanisms for the translation of these capped mRNAs without the requirement of eIF4E. eIF4G2, also known as death-associated protein 5 (DAP5), a homolog of eIFGI that lacks the eIF4E binding domain, utilizes eIF3d (a subunit of eIF3) to promote the translation of a subset of these mRNAs. Using fluorescence anisotropy-based equilibrium binding studies, we provide the first quantitative evidence of the recruitment of eIF3d as well as eIF3d and eIFG2 complexes to a subset of human mRNAs. Our quantitative studies demonstrate the critical role a fully methylated 50 mRNA cap structure plays in the recognition and recruitment of eIF3d, as well as the eIF3d and eIFG2 complex. By using luciferase reporter-based in vitro translation assays, we further show that cap-recognition ability correlates with the efficiency of translation of these mRNAs. Essentially, by preferably utilizing eIF3d and eIFG2, specific mRNA subsets are still able to translate in a cap-dependent manner even when eIF4E is sequestered. Our findings offer new insight into the use of eIF3d and eIF4G2 as an alternative for growth and survival under conditions of cellular stress. This novel mechanism of translation may offer new targets for therapeutic regulation of mRNA translation. 

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′UTR, and initiate cap-independent translation using elements called cap-independent translation enhancers or internal ribosome entry sites within their 5′UTRs. The interaction among initiation factors such as eukaryotic initiation factor 4E (eIF4E), eIF4A, and eIF4GI, especially in regulating the eIF4F complex during noncanonical translation initiation of different 5′UTR mRNAs, is poorly understood. Here, equilibrium-binding assays, CD studies and in vitro translation assays were used 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 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 used by cells to mitigate cellular stress conditions. 

Abstract Viral mRNAs that lack a 5′ m7GTP cap and a 3′ poly-A tail rely on structural elements in their untranslated regions (UTRs) to form unique RNA-protein complexes that regulate viral translation. Recent studies of the barley yellow dwarf virus (BYDV) have revealed eukaryotic initiation factor 3 (eIF3) plays a significant role in facilitating communication between its 5′ and 3′ UTRs by binding both UTRs simultaneously. This report uses in vitro translation assays, fluorescence anisotropy binding assays, and selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) footprinting to identify secondary structures that are selectively interacting with eIF3. SHAPE data also show that eIF3 alters its interaction with BYDV structures when another factor crucial for BYDV translation, eIF4F, is introduced by the 3′ BYDV translational enhancer (BTE). The observed BTE and eIF4F-induced shift of eIF3 position on the 5’ UTR and the translational effects of altering eIF3-binding structures (SLC and SLII) support a new model for BYDV translation initiation that requires the reorientation of eIF3 on BYDV UTRs. This eIF3 function in BYDV translation initiation is both reminiscent of and distinct from eIF3–RNA interactions found in other non-canonically translating mRNAs (e.g. HCV). This characterization of a new role in translation initiation expands the known functionality of eIF3 and may be broadly applicable to other non-canonically translating mRNAs.