Eukaryotic translation initiation factor 3 (eIF3) is the largest and most complex initiation factor first identified and purified from rabbit reticulocyte in 1976 (Benne and Hershey, 1976). eIF3 is involved in most reactions occurring in the initiation pathway, thereby or-ganizing a web of interactions between several translation initiation factors. In vertebrates and plants, the eIF3 complex consists of 13 nonidentical subunits, termed eIF3a-eIF3m and making up a mass of∼800kDa (Asanoet al., 1997; Browninget al., 2001). In budding yeast, the eIF3 complex is only made up by 5 subunits, orthologous to the mammalian subunits eIF3a, eIF3b, eIF3c, eIF3g and eIF3i, indicating a conserved core complex. In addition, budding yeast contains a protein orthologous to eIF3j, which is substochiometric and nonessential (Phan et al., 1998). However, not only the five eIF3 core subunits seem to be required for proper eIF3 function. Experiments in fission yeast showed that although eIF3f and eIF3m are not part of the conserved core complex, they are absolutely essential for viability (Akiyoshi et al., 2001).
1.1 Eukaryotic translation initiation 6
The first eIF3 subunit interaction studies were performedin vivo using budding yeast. By genomic deletion of predicted binding domains in tagged eIF3 subunits and subsequent determination of co-purified sub complexes, first models on yeast eIF3 subunit composi-tions and their interaccomposi-tions towards other initiation factors were reported (Phan et al., 1998; Valaseket al., 2002). Similar interactions were also shown in mammals including ad-ditional information on the interactions between core and non-core subunits, e.g. between eIF3b and eIF3e (Shalev et al., 2001).
Figure 1.2:Two distinct models on subunit composition of eIF3
Recently, two different subunit compositions for the humane eIF3 complex have been reported. (A)Zhouet al.
(2008) used a mass spectrometry approach to probe the subunit interactions within the complex. By “solution disruption experiments”, different subcomplexes were determined resulting in the shown model. The color code represents subunits containing PCI-domains (green), MPN-domains (red) or RNA-recognition motifs (yellow). (B) shows the subunit composition experimentally validated by Sunet al.(2011) and rephrased by Querol-Audiet al.
(2013). Sun and colleagues reconstituted the 13-subunit eIF3 complex inE.coliby using a stepwise assembly of co-expressed subcomplexes thereby mapping the interactions between the individual subunits. PCI-domain containing subunits are depicted in green, MPN-domain containing subunits are shown in red.
(adapted from Zhouet al., 2008; Querol-Audiet al., 2013)
Nowadays, two models on human eIF3 subunit composition are available as shown in figure 1.2. Zhou et al. (2008) analyzed a natively purified 13-subunit eIF3 complex by tandem mass spectrometry. By performing “solution disruption experiments”, they were able to detect three stable modules (eIF3(c-d-e-l-k), eIF3(f-h-m), eIF3(a-b-i-g)), which are brought together by interactions between subunits eIF3b and eIF3c and eIF3c and eIF3h.
Their model was further confirmed by immunoprecipitation experiments. The second composition is shown in figure 1.2B, based on a stepwise reconstitution of the human eIF3 complex inE.coli (Sunet al., 2011). The authors claim a stable 8-mer core comprised of the PCI/MPN subunits eIF3a, eIF3c, eIF3e, eIF3f, eIF3h, eIF3k, eIF3l and eIF3m.
The first 3D structure of human eIF3 was determined using complexes natively purified from HeLa cell lysate. At a resolution of 30 ˚A, a body-like shape for eIF3 was determined, showing a head domain, a left and right arm and left and right leg domain (Siridechadilok et al., 2005). Recent data at higher resolution reveal a more detailed view on the human
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eIF3 complex, however, the five extended domain shape remains as shown in figure 1.3A (Querol-Audi et al., 2013). Cryo electron microscopy was performed with reconstituted human eIF3 complexes, using the protocol published by Sunet al. (2011). This strategy enabled to add tags on certain subunits, thereby being able to determine the localization of individual subunits in the 3D model. eIF3h (marked in red) seems to be positioned in the center of the complex, which fits to the mass spectrometry data suggesting eIF3h to play a role in linking individual subcomplexes. Figure 1.3B shows the obtained eIF3 structure modeled onto the 40S ribosomal subunit. The location of the complex on 40S was previously suggested by Siridechadiloket al. (2005). The 3D model also contains the initiation factors eIF1 (blue) and eIF3 (yellow) that play a major role in correct AUG recognition during scanning of the mRNA by the 43S PIC.
Figure 1.3:3D structure of the human eIF3 complex
(A) By utilizing genetic tag visualization by electron microscopy at a resolution of 12 ˚A-16 ˚A, the location of the octameric eIF3 core subunits in the 13-subunit human eIF3 complex could be revealed (Querol-Audiet al., 2013).
(B)The 3D reconstitution of the eIF3 complex was modeled onto the 40S ribosomal subunit together with initiation factors eIF1(blue) and eIF1A (yellow). eIF3 was placed according to experimental data by Siridechadiloket al.
(2005), eIF1 and eIF1A due to crystal data (Rablet al., 2011). Potential localization of eIF3j is marked by the magenta dots.
(adopted from Querol-Audiet al., 2013)
As indicated earlier, eIF3 functions in various steps during translation initiation. These steps can be categorized as follows: (1) eIF3 acts as assembly platform for 43S PIC forma-tion. In yeast, eIF3 forms a multifactor complex (MFC) together with eIF1, eIF1A, eIF2 and eIF5. Thereby, eIF3 is the central factor in this complex holding it together. The MFC then binds the 40S ribosomal subunit in a cooperative manner (Asanoet al., 2000).
In mammals eIF3 stimulates binding of the ternary complex to the 40S ribosomal subunit,
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which is strongly impaired when eIF3b is mutated (Phan et al., 1998). In vitro studies further showed that the eIF3a-eIF3b-eIF3c subcomplex by itself can bind the 40S riboso-mal subunit and recruits the ternary complex (Phanet al., 2001). (2) eIF3 is essential for mRNA recruitment to the 43S PIC. Generally, eIF3 interacts with eIF4G, member of the cap binding eIF4F complex, thereby tethering the mRNA to the small ribosomal subunit (Korneeva et al., 2000). However, mRNA recruitment can also happen in an eIF4G inde-pendent manner. In yeast, some mRNAs can be directly recruited by the eIF3 complex;
in mammals, some mRNAs are attached by an interaction between eIF3 and eIF4E bound to the m7GTP cap (Jivotovskaya et al., 2006). (3) eIF3 is required for mRNA scanning and AUG initiation fidelity. eIF3c interacts with eIF5, the GTPase activating protein, and eIF1. When mutating eIF3c, binding of the other two translation initiation factors is impaired and a dramatic increase of initiation events at non-AUG (e.g. UUG) codons can be observed (Asano et al., 2000). The chain of interactions between eIF3, eIF1 and eIF5 seems to be crucial for proper accommodation of the initiator tRNA to the riboso-mal P-site. When eIF5 triggers GTPase activity on eIF2, eIF3 seems to slightly shifted thereby displacing eIF1, which is known to bind very close to the P-site. (4) eIF3 can induce reinitiation events and thereby plays a major role in gene-specific translational con-trol. In yeast eIF3 is required for reinitiating events when translating the GNC4 mRNA, in plants eIF3 is miss-used for repeating reinitiating events during the translation of a viral, polycistronic mRNA (Parket al., 2001). (5) eIF3 prevents rejoining of 40S and 60S.
Although the bulk mass of the eIF3 complex is though to bind to the solvent side of the 40S ribosomal subunit, an extended domain seems to bind to the interface surface thereby preventing and disrupting intersubunit binding (Siridechadilok et al., 2005). Thereby the eIF3 subunits eIF3d and eIF3j are though to play a major role in vertebrates, whereas subunits eIF3a and eIF3c are required for 40S binding in yeast (Fraseret al., 2004; Nielsen et al., 2006).
Although much seems to be known about the function and interactions of and within the eIF3 complex, many details remain unclear. So far, mainly human and budding yeast eIF3 complexes were analyzed; the plant complex however remains greatly unstudied. It is known that eIF3 in plants also contain 13 subunits, however the arrangement of the subunits in the complex and their interaction within the complex are unknown and there might be further eIF3 subunits or isoforms so far not identified.
Recent studies showed that the human eIF3 complex can be reconstituted using either the Bacoluvirus system (Masutaniet al., 2007) or evenE.coli (Sunet al., 2011). These major breakthroughs enable a new level of analyzing this large factor by introducing mutations or deletions that would lead to a lethal phenotype in in vivo experiments. Recombinant