Toward the mechanism of eIF4F-mediated ribosomal attachment to mammalian capped mRNAs

Abstract

Ribosomal attachment to mammalian capped mRNAs is achieved through the cap-eukaryotic initiation factor 4E (eIF4E)-eIF4G-eIF3-40S chain of interactions, but the mechanism by which mRNA enters the mRNA-binding channel of the 40S subunit remains unknown. To investigate this process, we recapitulated initiation on capped mRNAs in vitro using a reconstituted translation system. Formation of initiation complexes at 5 ′ -terminal AUGs was stimulated by the eIF4E-cap interaction and followed "the first AUG" rule, indicating that it did not occur by backward scanning. Initiation complexes formed even at the very 5 ′ end of mRNA, implying that Met-tRNA i Met inspects mRNA from the first nucleotide and that initiation does not have a "blind spot." In assembled initiation complexes, the cap was no longer associated with eIF4E. Omission of eIF4A or disruption of eIF4E-eIF4G-eIF3 interactions converted eIF4E into a specific inhibitor of initiation on capped mRNAs. Taken together, these results are consistent with the model in which eIF4E-eIF4G-eIF3-40S interactions place eIF4E at the leading edge of the 40S subunit, and mRNA is threaded into the mRNA-binding channel such that Met-tRNA i Met can inspect it from the first nucleotide. Before entering, eIF4E likely dissociates from the cap to overcome steric hindrance. We also found that the m 7 G cap specifically interacts with eIF3l.[Keywords: eukaryotic translation initiation; eIF4E; eIF4F; m 7 G; eIF3l; 40S ribosomal subunit]Supplemental material is available for this article.Received April 7, 2016; revised version accepted June 1, 2016.Translation initiation on the majority of mammalian cellular mRNAs occurs by the scanning mechanism (Jackson et al. 2010). First, eukaryotic initiation factor 2 (eIF2), GTP, and Met-tRNA i Met form a ternary complex (eIF2-TC), which, with the multisubunit eIF3 and monomeric eIF1 and eIF1A, binds to the 40S ribosomal subunit, yielding a 43S preinitiation complex. Attachment of the 43S complex to capped mRNA is mediated by eIF4A, eIF4B, and eIF4F. eIF4F comprises the cap-binding protein eIF4E, the DEAD-box RNA helicase eIF4A (which also exists in a free form), and the eIF4G scaffold, which interacts with the other two subunits as well as eIF3. eIF4A's helicase activity is stimulated by both eIF4G and eIF4B. Group 4 eIFs cooperatively unwind the cap-proximal region of mRNA, preparing it for attachment of 43S complexes, which is promoted by the eIF4G-eIF3 interaction. After attachment, 43S complexes scan to the first AUG codon in a favorable nucleotide context, where they form 48S initiation complexes with established codonanti-codon base-pairing. Group 4 eIFs also assist 43S complexes during scanning. Scanning on mRNAs with highly structured 5 ′ untranslated regions (UTRs) additionally requires DHX29, a DExH-box protein that interacts directly with 40S subunits. eIF1, in cooperation with eIF1A, ensures the fidelity of initiation codon selection, discriminating against initiation at non-AUG codons and AUGs that are too close to the 5 ′ end of mRN...

Molecular Landscape of the Ribosome Pre-initiation Complex during mRNA Scanning: Structural Role for eIF3c and Its Control by eIF5

“…The other four factors engage in numerous mutual interactions to form the multifactor complex (MFC) with Met-tRNA i Met bound to eIF2-GTP in the ternary complex, whereby MFC can be isolated free of ribosomes from various eukaryotes (Asano et al, 2000; Dennis et al, 2009; Meleppattu et al, 2015; Sokabe et al, 2012). eIF4F, comprised of m 7 G-cap binding subunit eIF4E, RNA helicase eIF4A and scaffold eIF4G, mediates attachment of the mRNA 5′ end to the PIC in its open, scanning-competent conformation (Kumar et al, 2016). A key event in start codon selection is dissociation from the 40S of eIF1, a gatekeeper molecule that maintains the open conformation of the PIC (Pestova and Kolupaeva, 2002; Saini et al, 2010).…”

Regulation of translation initiation factor eIF2B at the hub of the integrated stress response

“…(b) Cartoon image indicating major protein interaction partners important in the ISR eIFs 4A and 4B help eliminate mRNA secondary structure to promote PIC-mRNA interactions (Hinnebusch, 2014;Merrick & Pavitt, 2018). Recent evidence from in vitro reactions suggests that the PIC is threaded on to the mRNA so that tRNA i Met can recognize AUG codons close to the 5 0 cap (Kumar, Hellen, & Pestova, 2016). From here, eIFs 1, 1A, 3, 4A, and 4B promote "scanning" of the PIC along the mRNA 5 0 leader seeking an AUG codon in an appropriate context for base-pairing and initiation.…”

Structural basis for LeishIF4E-1 modulation by an interacting protein in the human parasite Leishmania major

“…In higher eukaryotes, the translational initiation complex assembles at the m 7 GTP cap of messenger RNAs (mRNAs) (9) through the eukaryotic initiation factor 4F complex (eIF4F) (10). eIF4F comprises the cap-binding protein eIF4E, the DEAD-box RNA helicase eIF4A, and the scaffold protein eIF4G (11,12).…”