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

Abstract

Background: AUG recognition is promoted by several initiation factors (eIFs).Results: eIF5 interacts with the extreme N terminus of eIF3c/Nip1 to promote pre-initiation complex assembly, and eIF1 binds the region that immediately follows.Conclusion: eIF1 binding to c/Nip1 is equally important for its 40 S ribosome recruitment and AUG selection.Significance: Understanding start codon selection that sets the reading frame for decoding is key in gene expression studies.

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

“…Other studies identified interactions between the eIF3a NTD and uS2 (Valášek et al, 2003; Kouba et al, 2012b), the eIF3c CTD and RACK1 (Kouba et al, 2012a), and between the eIF3c NTD and both eIF5 and eIF1 (Asano et al, 2000; Valasek et al, 2004). This final observation, together with the discovery of mutations in the eIF3c NTD that affect the fidelity of start-codon recognition (Valasek et al, 2004; Karásková et al, 2012), supports the putative identification of regions of density near the P site in the py48S-closed structure (Llácer et al, 2015). …”

“…Genetic and biochemical evidence indicate that eIF3 stabilizes both the 43S (Asano et al, 2000; Kolupaeva et al, 2005; Maag et al, 2005; Valášek et al, 2003) and 48S PIC (Chiu et al, 2010; Khoshnevis et al, 2014; Phan et al, 2001) and interacts with TC (Valášek et al, 2002), eIF1 (Fletcher et al, 1999; Valasek et al, 2004), eIF1A (Olsen et al, 2003), and eIF5 (Asano et al, 2001; Phan et al, 1998), as well as with the 40S subunit near both the mRNA entry and exit channels (Kouba et al, 2012a, 2012b; Pisarev et al, 2008; Valášek et al, 2003). eIF3 also plays roles in loading the mRNA onto the PIC (Jivotovskaya et al, 2006; Mitchell et al, 2010; Pestova and Kolupaeva, 2002) and in scanning of the mRNA to locate the start codon (Chiu et al, 2010; Cuchalova et al, 2010; Karásková et al, 2012; Nielsen et al, 2006; Valasek et al, 2004). How eIF3 contributes to these diverse events is not yet clear.…”

“…To interrogate the mechanistic roles of the domains of eIF3, we purified a library of S. cerevisiae eIF3 variants – all of which were previously characterized in vivo (Chiu et al, 2010; Cuchalova et al, 2010; Herrmannová et al, 2012; Karásková et al, 2012; Khoshnevis et al, 2014; Kouba et al, 2012a; Nielsen et al, 2004, 2006; Szamecz et al, 2008) – to probe their effects on steps and interactions in the translation initiation pathway in an in vitro - reconstituted yeast translation system (Acker et al, 2007; Algire et al, 2002; Mitchell et al, 2010). This library contains variants with mutations spanning the five essential subunits of eIF3 (Table 1).…”

Rps5-Rps16 communication is essential for efficient translation initiation in yeastS. cerevisiae

“…Sui − mutants increase initiation at the UUG codon encoding the third amino acid in the His4 protein, thus bypassing the requirement for the AUG start codon in HIS4 mRNA for yeast growth on medium lacking histidine (37). Extensive analysis of Sui − mutations in eIF1 has established that they elevate aberrant initiation at UUG codons by reducing the recruitment of eIF1, or enabling its inappropriate release from the 48S PIC at non-AUG codons, owing to weakened interactions of eIF1 mutants with the 40S subunit or MFC components (reviewed in (31) (38,39). It was further suggested that gain-of-function Sui − mutations in eIF2β or eIF5 that accelerate GTP hydrolysis by eIF2 and induce premature release of Met-tRNA i Met from eIF2·GDP on the scanning 40S subunit enable ribosomes to initiate translation from the UUG codon in HIS4 mRNA (13).…”

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.…”