The C-Terminal Region of Eukaryotic Translation Initiation Factor 3a (eIF3a) Promotes mRNA Recruitment, Scanning, and, Together with eIF3j and the eIF3b RNA Recognition Motif, Selection of AUG Start Codons

Abstract

The C-terminal domain (CTD) of the a/Tif32 subunit of budding yeast eukaryotic translation initiation factor 3 (eIF3) interacts with eIF3 subunits j/Hcr1 and b/Prt1 and can bind helices 16 to 18 of 18S rRNA, suggesting proximity to the mRNA entry channel of the 40S subunit. We have identified substitutions in the conserved Lys-Glu-Arg-Arg (KERR) motif and in residues of the nearby box6 element of the a/Tif32 CTD that impair mRNA recruitment by 43S preinitiation complexes (PICs) and confer phenotypes indicating defects in scanning and start codon recognition. The normally dispensable CTD of j/Hcr1 is required for its binding to a/Tif32 and to mitigate the growth defects of these a/Tif32 mutants, indicating physical and functional interactions between these two domains. The a/Tif32 CTD and the j/Hcr1 N-terminal domain (NTD) also interact with the RNA recognition motif (RRM) in b/Prt1, and mutations in both subunits that disrupt their interactions with the RRM increase leaky scanning of an AUG codon. These results, and our demonstration that the extreme CTD of a/Tif32 binds to Rps2 and Rps3, lead us to propose that the a/Tif32 CTD directly stabilizes 43S subunit-mRNA interaction and that the b/Prt1-RRM-j/ Hcr1-a/Tif32-CTD module binds near the mRNA entry channel and regulates the transition between scanningconducive and initiation-competent conformations of the PIC.Eukaryotic translation initiation factor 3 (eIF3) is a multisubunit protein complex that has been implicated in several steps of the translation initiation pathway (reviewed in reference 19). These steps include recruitment of the eIF2-GTP-Met-tRNA i Met ternary complex (TC) and other eIFs to the small (40S) ribosomal subunit to form the 43S preinitiation complex (PIC), mRNA recruitment by the 43S PIC, and subsequent scanning of the 5Ј untranslated region (UTR) for an AUG start codon. The eIF3 in the budding yeast Saccharomyces cerevisiae is composed of only 6 subunits (a/Tif32, b/Prt1, c/Nip1, i/Tif34, g/Tif35, and j/Hcr1), which have homologs in the larger, 13-subunit eIF3 complex in mammals. Yeast eIF3 can be purified with the TC, eIF1, and eIF5 in a ribosome-free assembly called the multifactor complex (MFC) (2), whose formation appears to promote assembly or stability of the 43S PIC and to stimulate scanning and AUG selection (10,23,32,42,48,49,51).In mammals, there is evidence that eIF3 enhances recruitment of mRNA by interacting directly with eIF4G, the "scaffold" subunit of mRNA cap-binding complex eIF4F, and forming a protein bridge between mRNA and the 43S PIC (24,25,35). In budding yeast, direct eIF3-eIF4G interaction has not been detected, and the eIF3-binding domain (25) is not evident in yeast eIF4G. Moreover, depletion of eIF3, but not eIF4G, from yeast cells provokes a strong decrease in the amount of an mRNA (RPL41A) associated with native PICs (23). However, since depletion of eIF3 also reduced the amounts of other MFC components associated with PICs, it remained unclear whether eIF3 acts directly in mRNA recruitment. In favor of a dire...

Functional reconstitution of human eukaryotic translation initiation factor 3 (eIF3)

“…4) (24) suggests that eIF3 may envelope the 40S ribosomal subunit, using flexible regions emanating from the PCI/MPN core in the assembly of preinitiation and initiation complexes (24,(37)(38)(39)(40). Genetic and biochemical experiments identified interactions between subunits eIF3a and eIF3c and the solvent side of the 40S subunit (37,38). The flexible C terminus of eIF3a is located near the mRNA entry tunnel, whereas the N terminus of eIF3c is likely positioned near the 40S subunit platform where it interacts with eIF1 and eIF5 situated at the subunit interface (41).…”

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…Spontaneous Sui 2 mutations that enhance initiation from a UUG codon were isolated in eIF1 (Yoon and Donahue 1992), all three subunits of eIF2 Cigan et al 1989;Huang et al 1997), and in eIF5 (encoded by TIF5) (Huang et al 1997). In subsequent directed screens Sui 2 mutations have also been isolated in eIF1A ), eIF3 subunits Chiu et al 2010;Elantak et al 2010;Karaskova et al 2012), and in 18S rRNA (Nemoto et al 2010). In contrast to the Sui 2 mutations, which relax the stringency for start codon selection, a second class of mutations enhances start codon selectivity.…”

Molecular Mechanism of Scanning and Start Codon Selection in Eukaryotes

“…The a/Tif32 KERR motif and box6 substitutions mentioned above also confer Ssu Ϫ phenotypes and reduce the efficiency of scanning through a long 5ЈUTR in yeast cells. The box6 substitution also impairs scanning through a stem-loop located far downstream from the 5Ј end of a reporter mRNA (33). Considering that the a/Tif32 CTD interacts directly with 40S structural elements (h16 and Rps3e) that promote the open conformation of the mRNA channel latch (159), it was suggested that these mutations might impair the opening of the latch, although they could also reduce the function of a helicase at the entry channel that removes the secondary structure ahead of the scanning ribosome.…”

“…Substitutions in conserved residues in the C-terminal region of yeast eIF3a/Tif32 (the "KERR motif" and "box6") impair the binding of native mRNAs to 43S PICs in vivo without reducing the 40S binding of the TC or any other 43S components (33 capped mRNAs in vitro, being more critically required than eIF4G (139). Consistent with the prediction that eIF3 extends the mRNA exit channel (173), yeast eIF3 more strongly enhanced 43S binding to a model mRNA with a long leader upstream of AUG (that would protrude from the exit channel) than to one containing a short leader but a long 3Ј extension (that would protrude from the entry channel) (139).…”