Structural integrity of the PCI domain of eIF3a/TIF32 is required for mRNA recruitment to the 43S pre-initiation complexes

Abstract

Transfer of genetic information from genes into proteins is mediated by messenger RNA (mRNA) that must be first recruited to ribosomal pre-initiation complexes (PICs) by a mechanism that is still poorly understood. Recent studies showed that besides eIF4F and poly(A)-binding protein, eIF3 also plays a critical role in this process, yet the molecular mechanism of its action is unknown. We showed previously that the PCI domain of the eIF3c/NIP1 subunit of yeast eIF3 is involved in RNA binding. To assess the role of the second PCI domain of eIF3 present in eIF3a/TIF32, we performed its mutational analysis and identified a 10-Ala-substitution (Box37) that severely reduces amounts of model mRNA in the 43–48S PICs in vivo as the major, if not the only, detectable defect. Crystal structure analysis of the a/TIF32-PCI domain at 2.65-Å resolution showed that it is required for integrity of the eIF3 core and, similarly to the c/NIP1-PCI, is capable of RNA binding. The putative RNA-binding surface defined by positively charged areas contains two Box37 residues, R363 and K364. Their substitutions with alanines severely impair the mRNA recruitment step in vivo suggesting that a/TIF32-PCI represents one of the key domains ensuring stable and efficient mRNA delivery to the PICs.

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

“…The eIF3a N-terminal domain (NTD) interacts functionally with mRNA near the exit channel, enhancing reinitiation upon translation of the upstream open reading frame 1 (uORF1) and uORF2 of GCN4 mRNA in a manner dependent on the sequence upstream of these two uORFs (Gunišová et al, 2016; Gunišová and Valášek, 2014; Munzarová et al, 2011; Szamecz et al, 2008). Consistent with this, mammalian eIF3 can be cross-linked to mRNA at positions 8–17 nucleotides upstream of the AUG codon (Pisarev et al, 2008), near the exit channel, and a mutation within the eIF3a NTD, which has been located near the exit channel in structures of the PIC, interferes with mRNA recruitment in yeast cells (Khoshnevis et al, 2014). …”

“…A sixth nonessential and nonstoichiometric subunit, eIF3j, associates loosely with the complex (Elantak et al, 2010; Fraser et al, 2004; Nielsen et al, 2006; Valasek et al, 2001a), though its role during initiation remains unclear (Beznosková et al, 2013; Fraser et al, 2007; Mitchell et al, 2010). 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).…”

“…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).…”

Functional and Biochemical Characterization of Human Eukaryotic Translation Initiation Factor 3 in Living Cells

“…Among a very few examples of characterized interactions, two recently identified RNA binding domains of eIF3a and eIF3c were shown to mediate eIF3 binding to the 40S subunit and to the HCV mRNA internal ribosome entry site (IRES) and thus promote HCV translation (7). It is highly likely that under standard conditions, these two subunits may promote mRNA recruitment to the 43S PICs, as was proposed for their yeast counterparts TIF32 and NIP1 (21–23). Interestingly, eIF3a and eIF3c were also proposed to interact with eIFs 1 and 1A (14) (Fig.…”

The role of eIF3 and its individual subunits in cancer

“…The recently solved structure of the human COP9 PCI/MPN octameric complex at 3.8 Å also depicts a 5-appendage structure and provides detailed insight into how the two MPN proteins interact with the 6 PCI proteins [57]. Atomicresolution X-ray crystallographic structures of individual eIF3 subunits or their fragments have been reported for: human 3 k [58]; yeast 3i in complex with a C-terminal fragment of yeast 3b [59]; the central WD40 domain of 3b from Chaetomium thermophilum [60] and yeast [47]; a complex of human 3j and the N-terminal region of 3b [61]; the RRM region of yeast eIF3b [62]; the PCI domain of yeast 3a [63]; and a complex of the PCI domains of yeast 3a and 3c [47]. Combining information derived from subunit atomic structures, cryo-electron microscopy and crosslinking data, the Ban group [47] recently has modeled both yeast and mammalian eIF3 on the surface of the 40S ribosomal subunit, thereby identifying where individual eIF3 subunits bind (Fig.…”