Functional Characterization of the Role of the N-terminal Domain of the c/Nip1 Subunit of Eukaryotic Initiation Factor 3 (eIF3) in AUG Recognition

Martina Karásková,Stanislava Gunišová,Anna Herrmannová,Susan Wagner,Vanda Munzarová,Leoš Shivaya Valášek

doi:10.1074/jbc.m112.386656

Abstract

In eukaryotes, for a protein to be synthesized, the 40 S subunit has to first scan the 5'-UTR of the mRNA until it has encountered the AUG start codon. Several initiation factors that ensure high fidelity of AUG recognition were identified previously, including eIF1A, eIF1, eIF2, and eIF5. In addition, eIF3 was proposed to coordinate their functions in this process as well as to promote their initial binding to 40 S subunits. Here we subjected several previously identified segments of the N-terminal domain (NTD) of the eIF3c/Nip1 subunit, which mediates eIF3 binding to eIF1 and eIF5, to semirandom mutagenesis to investigate the molecular mechanism of eIF3 involvement in these reactions. Three major classes of mutant substitutions or internal deletions were isolated that affect either the assembly of preinitiation complexes (PICs), scanning for AUG, or both. We show that eIF5 binds to the extreme c/Nip1-NTD (residues 1-45) and that impairing this interaction predominantly affects the PIC formation. eIF1 interacts with the region (60-137) that immediately follows, and altering this contact deregulates AUG recognition. Together, our data indicate that binding of eIF1 to the c/Nip1-NTD is equally important for its initial recruitment to PICs and for its proper functioning in selecting the translational start site.

Highlights

AUG recognition is promoted by several initiation factors
We show that eIF5 binds to the extreme c/Nip1-N-terminal domain (NTD) and that impairing this interaction predominantly affects the preinitiation complexes (PICs) formation. eIF1 interacts with the region (60 –137) that immediately follows, and altering this contact deregulates AUG recognition
By subjecting the N-terminal 160 amino acids of c/Nip1 to the clustered 10-alanine mutagenesis, we showed previously that these c/Nip1 interactions stimulate the assembly of the 43 S PICs and somehow coordinate the functions of eIF1 and eIF5 in stringent AUG selection [11]

Summary

Background

AUG recognition is promoted by several initiation factors (eIFs). Results: eIF5 interacts with the extreme N terminus of eIF3c/Nip to promote pre-initiation complex assembly, and eIF1 binds the region that immediately follows. That requires dissolving the latch formed by helices 18 and 34 of 18 S rRNA and establishing a new interaction between Rps and helix 16 [2] This so-called open/scanning-conducive conformation with the anticodon of MettRNAiMet not fully engaged in the ribosomal P-site to prevent premature engagement with putative start codons is maintained during scanning for the AUG start codon in an ATP-dependent process Some of the identified mutations affected assembly of the PICs. increased frequency of skipping the AUG of uORF1 in the GCN4-lacZ reporter (a leaky scanning phenotype) was observed with mutations disrupting the web of mutual interactions among the members of an eIF3 module composed of the RNA recognition motif in the NTD of b/Prt and j/Hcr and the C-terminal domain (CTD) of a/Tif (Fig. 1) [9, 10]. We propose a model suggesting that the productive recruitment of eIF1 to 40 S ribosomes and its proper functioning during the AUG recognition process depends on its contact with the stretch of amino acid residues 60 –137 of the c/Nip1-NTD

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION