Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

Abstract

Eukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2•GTP•Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncovered a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA.DOI: http://dx.doi.org/10.7554/eLife.20934.001

Rps3/uS3 promotes mRNA binding at the 40S ribosome entry channel and stabilizes preinitiation complexes at start codons

“…7B, cyan bars), which lack mRNA bases at the entry channel but fully occupy and protrude from the exit channel. These last findings are consistent with our previous observation that interactions at the entry channel are redundant with those at the exit channel (18), and also suggest that R116D does not impair other aspects of 40S structure or function that would compromise stable 48S PIC assembly with these mRNAs beyond the loss of mRNA-40S interactions at the entry channel.…”

“…Fig. 7 B and C, 5′5 mRNA, gray bars) (18). The fact that mRNA nucleotides at the entry channel can compensate for the absence of eIF3-mediated PIC-mRNA interactions at the exit channel implies the existence of compensatory PIC-mRNA interactions at the entry channel.…”

“…A second contact occurs near the entry channel and involves segments of the eIF3a, b, g, and i subunits; at least some of these interactions appear to be dynamic, as alternative contacts with eIFs anchored in the decoding center have also been observed (20)(21)(22). We recently presented evidence that the PCI domain of eIF3a mediates a key stabilizing interaction between the PIC and mRNA at the exit channel (18). Using a panel of m 7 G-capped, unstructured model mRNAs to reconstitute 48S PICs, we determined that the eIF3a PCI domain Fig.…”

“…We have shown that the β-hairpin of yeast Rps5 is important for both efficient and accurate translation initiation in vivo and the stability of P IN complexes reconstituted in vitro (14). Approximately 7 additional mRNA nucleotides upstream of the −3 position occupy the 40S exit channel, and there is evidence that these 40S-mRNA interactions plus additional contacts between segments of eIF3 and mRNA nucleotides protruding from the exit channel also enhance PIC assembly at the start codon (15)(16)(17)(18).…”

“…We recently analyzed the role of eIF3 subunits and domains in mediating stable interactions of the PIC with mRNA nucleotides located at the entry or exit channel of the 40S subunit by reconstituting PICs with different model mRNAs designed to incompletely occupy either the entry or exit channel of the 40S subunit (18). Capped model mRNAs designated "5′5-AUG" and "5′11-AUG" contain only 5 or 11 nt located 5′ of the AUG start codon, respectively, but >30 nt 3′ of the AUG.…”