Abstract
Translation re-initiation provides the molecular basis for translational control of mammalian ATF4 and yeast GCN4 mediated by short upstream open reading (uORFs) in response to eIF2 phosphorylation. eIF4G is the major adaptor subunit of eIF4F that binds the cap-binding subunit eIF4E and the mRNA helicase eIF4A and is also required for re-initiation in mammals. Here we show that the yeast eIF4G2 mutations altering eIF4E- and eIF4A-binding sites increase re-initiation at GCN4 and impair recognition of the start codons of uORF1 or uORF4 located after uORF1. The increase in re-initiation at GCN4 was partially suppressed by increasing the distance between uORF1 and GCN4, suggesting that the mutations decrease the migration rate of the scanning ribosome in the GCN4 leader. Interestingly, eIF4E overexpression suppressed both the phenotypes caused by the mutation altering eIF4E-binding site. Thus, eIF4F is required for accurate AUG selection and re-initiation also in yeast, and the eIF4G interaction with the mRNA-cap appears to promote eIF4F re-acquisition by the re-initiating 40 S subunit. However, eIF4A overexpression suppressed the impaired AUG recognition but not the increase in re-initiation caused by the mutations altering eIF4A-binding site. These results not only provide evidence that mRNA unwinding by eIF4A stimulates start codon recognition, but also suggest that the eIF4A-binding site on eIF4G made of the HEAT domain stimulates the ribosomal scanning independent of eIF4A. Based on the RNA-binding activities identified within the unstructured segments flanking the eIF4G2 HEAT domain, we discuss the role of the HEAT domain in scanning beyond loading eIF4A onto the pre-initiation complex.
Highlights
The mRNA interacts with the 40 S subunit over the specific path on the 40 S subunit that includes the decoding sites located between its head and body/platform domains (3). eIF4F and the attendant eIF4A-dependent unwinding remove the secondary structure for a sufficient stretch of the mRNA 5Ј region to allow stable formation of the 48 S PIC at the mRNA 5Ј-end (4)
Short upstream ORF (uORF) in rabbit reticulocyte lysates requires eIF4F that has We previously showed that tif4632-1 altering the eIF4G2 been loaded onto the mRNA by translation of the uORF, dem- HEAT1 weakly but significantly increases the frequency of onstrating that eIF4F is directly involved in scanning (13)
As scanning of uORF1 (Fig. 3B), much larger increase of Ͼ1000 shown in Fig. 5B, panel 1, we found that the magnitude of units observed with pM199 in these mutants suggests that the increase in GCN4-lacZ expression from pA77, with a wider increase is not due to the leaky scanning mechanism. intergenic distance, by each of the tif4632 mutations tested was Rather, the tif4632 mutations appear to increase the frequency significantly lower than that of increase in expression from of translational re-initiation following uORF1
Summary
Recent studies propose that this eIF5-stimulated GTP hydrolysis occurs at an earlier stage of PIC formation, with the resulting GDP and Pi remaining on eIF2, until AUG is encountered by the anticodon of Met-tRNAiMet located at the 40 S P-site (7). In response to the anticodon pairing to the AUG, the release of eIF1 is proposed to trigger the transition to the closed conformation, thereby allowing Pi to dissociate from eIF2 (10) These events lead to the dissociation of eIF2-GDP, eIF5, and perhaps other eIFs, which otherwise prevent binding of eIF5B and the eIF5B-mediated 60 S subunit joining that results in formation of the 80 S IC (11). ORF is activated in response to amino acid starvation and the attendant Gcn2p-catalyzed phosphorylation of eIF2 (14) This unique translational regulation is mediated by uORFs present in the leader region of GCN4 mRNA
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