RNA Recognition Motif 2 of Yeast Pab1p Is Required for Its Functional Interaction with Eukaryotic Translation Initiation Factor 4G

The eukaryotic mRNA 3′ poly(A) tail and its associated poly(A)-binding protein (Pab1p) are important regulators of gene expression. One role for this complex in the yeast Saccharomyces cerevisiae is in translation initiation through an interaction with a 115-amino-acid region of the translation initiation factor eIF4G. The eIF4G-interacting domain of Pab1p was mapped to its second RNA recognition motif (RRM2) in an in vitro binding assay. Moreover, RRM2 of Pab1p was required for poly(A) tail-dependent translation in yeast extracts. An analysis of a site-directed Pab1p mutation which bound to eIF4G but did not stimulate translation of uncapped, polyadenylated mRNA suggested additional Pab1p-dependent events during translation initiation. These results support the model that the association of RRM2 of yeast Pab1p with eIF4G is a prerequisite for the poly(A) tail to stimulate the translation of mRNA in vitro.     

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

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 scanning-conducive 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-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 5′ 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 direct role for eIF3, cross-linking analysis of reconstituted mammalian 48S PICs identified contacts of subunits eIF3a and eIF3d with mRNA residues 8 to 17 nucleotides (nt) upstream of the AUG codon, suggesting that these subunits form an extension of the mRNA exit channel (37). Consistent with this, we found that the N-terminal domain (NTD) of yeast a/Tif32 binds Rps0A, located near the mRNA exit pore, and functionally interacts with sequences 5′ to the regulatory upstream open reading frame 1 (uORF1) in GCN4 mRNA (42). Despite these advances, in vivo evidence supporting a direct role of eIF3 in mRNA recruitment by 43S PICs is lacking.Recently, there has been progress in elucidating the molecular mechanisms involved in ribosomal scanning and AUG selection. Reconstituted mammalian 43S PICs containing only eIF1, -1A, and -3 and the TC can scan the leader of an unstructured message and form a stable 48S PIC at the 5′-proximal AUG codon (35). eIF1 and -1A are thought to promote scanning by stabilizing an open conformation of the 40S subunit (6, 13, 26, 27), which appears to involve opening the “latch” on the mRNA entry channel formed by helices 18 and 34 of 18S rRNA (33). eIF1A also promotes a mode of TC binding conducive to scanning (39) and seems to prevent full accommodation of Met-in the P site at non-AUG codons (53). The GTP bound to eIF2 is hydrolyzed, in a manner stimulated by eIF5, but release of phosphate (P_i) from eIF2-GDP-P_i is blocked by eIF1 (1). Entry of AUG into the P site triggers relocation of eIF1 from its binding site on the 40S subunit (27), allowing P_i release (1) and stabilizing the closed, scanning-arrested conformation of the 40S subunit (33).Mutations in eIF1 and eIF1A that reduce the stringency of start codon recognition have been isolated by their ability to increase initiation at a UUG codon in his4 alleles lacking the AUG start codon (the Sui⁻ phenotype) (6, 12, 13, 29, 38, 39, 52). eIF1A mutations with the opposite effect of lowering UUG initiation in the presence of a different Sui⁻ mutation (the Ssu⁻ phenotype) were also obtained (13, 39). Previously, we identified Sui⁻ and Ssu⁻ mutations in the N-terminal domain of eIF3 subunit c/Nip1, which alter its contacts with eIF1, -2, and -5, suggesting that integrity of the MFC is important for the accuracy of AUG selection (49).Several genetic findings also implicate eIF3 in the efficiency of scanning and AUG recognition. The prt1-1 point mutation in b/Prt1 (S518F) (11) impairs translational control of GCN4 mRNA in a manner suggesting a reduced rate of scanning between the short uORFs involved in this control mechanism (30). Disrupting an interaction between a hydrophobic pocket of the noncanonical RNA recognition motif (RRM) in the N terminus of b/Prt1 (henceforth referred to as b/RRM) and a Trp residue in the N-terminal acidic motif of j/Hcr1 (Trp-37) severely reduces the efficiency of initiation at the AUG of uORF1 in GCN4 mRNA, the phenomenon of leaky scanning, implicating the connection between the b/RRM and j/Hcr1 NTD (henceforth referred to as j/NTD) in efficient AUG recognition (10). Similarly, a multiple Ala substitution in RNP1 of the b/RRM evoked leaky scanning of the AUG codon of GCN4 uORF1 (uAUG-1) (32).Interestingly, besides the b/RRM-j/NTD contact, the b/RRM can simultaneously bind to the j/Hcr1-like domain (HLD) in a/Tif32, and j/Hcr1 also independently binds a/Tif32 (50). This network of interactions involving the b/RRM, a/Tif32-HLD, and j/Hcr1 segments was shown to stabilize an eIF3 subassembly (50), referred to below as the b/RRM-j/Hcr1-a/Tif32-CTD module; however, it was not known whether the a/Tif32 HLD component of this module also participates in AUG recognition or other specific steps of initiation.In this report, we provide evidence that the evolutionarily conserved KERR motif in the a/Tif32 HLD (hereafter referred to as a/HLD) functions to enhance mRNA recruitment by 43S PICs, processivity of scanning, and the efficiency of AUG recognition. The identification of Ssu⁻ phenotypes for both KERR mutations and replacement of a nearby element (box6) further implicates the a/HLD in promoting the closed, scanning-arrested conformation of the PIC at start codons. Combining these results with our finding that the a/Tif32 CTD binds the 40S proteins Rps3 and Rps2 and the recent evidence that j/Hcr1 promotes AUG recognition and binds Rps2 leads us to propose that the a/HLD is positioned near the 40S mRNA entry channel, where it promotes mRNA binding and, together with j/Hcr1 and the b/RRM, modulates the transition between the open and closed conformations of the PIC during scanning and AUG recognition.     

Crystal Structure of the RNA Recognition Motif of Yeast Translation Initiation Factor eIF3b Reveals Differences to Human eIF3b

Background

The multi-subunit eukaryotic initiation factor3 (eIF3) plays a central role in the initiation step of protein synthesis in eukaryotes. One of its large subunits, eIF3b, serves as a scaffold within eIF3 as it interacts with several other subunits. It harbors an RNA Recognition Motif (RRM), which is shown to be a non-canonical RRM in human as it is not capable to interact with oligonucleotides, but rather interacts with eIF3j, a sub-stoichiometric subunit of eIF3.

Principal Finding

We have analyzed the high-resolution crystal structure of the eIF3b RRM domain from yeast. It exhibits the same fold as its human ortholog, with similar charge distribution on the surface interacting with the eIF3j in human. Thermodynamic analysis of the interaction between yeast eIF3b-RRM and eIF3j revealed the same range of enthalpy change and dissociation constant as for the human proteins, providing another line of evidence for the same mode of interaction between eIF3b and eIF3j in both organisms. However, analysis of the surface charge distribution of the putative RNA-binding β-sheet suggested that in contrast to its human ortholog, it potentially could bind oligonucleotides. Three-dimensional positioning of the so called “RNP1” motif in this domain is similar to other canonical RRMs, suggesting that this domain might indeed be a canonical RRM, conferring oligonucleotide binding capability to eIF3 in yeast. Interaction studies with yeast total RNA extract confirmed the proposed RNA binding activity of yeast eIF3b-RRM.

Conclusion

We showed that yeast eIF3b-RRM interacts with eIF3j in a manner similar to its human ortholog. However, it shows similarities in the oligonucleotide binding surface to canonical RRMs and interacts with yeast total RNA. The proposed RNA binding activity of eIF3b-RRM may help eIF3 to either bind to the ribosome or recruit the mRNA to the 43S pre-initiation complex.     

Eukaryotic Translation Initiation Factor 4AIII (eIF4AIII) Is Functionally Distinct from eIF4AI and eIF4AII

Eukaryotic initiation factor 4A (eIF4A) is an RNA-dependent ATPase and ATP-dependent RNA helicase that is thought to melt the 5' proximal secondary structure of eukaryotic mRNAs to facilitate attachment of the 40S ribosomal subunit. eIF4A functions in a complex termed eIF4F with two other initiation factors (eIF4E and eIF4G). Two isoforms of eIF4A, eIF4AI and eIF4AII, which are encoded by two different genes, are functionally indistinguishable. A third member of the eIF4A family, eIF4AIII, whose human homolog exhibits 65% amino acid identity to human eIF4AI, has also been cloned from Xenopus and tobacco, but its function in translation has not been characterized. In this study, human eIF4AIII was characterized biochemically. While eIF4AIII, like eIF4AI, exhibits RNA-dependent ATPase activity and ATP-dependent RNA helicase activity, it fails to substitute for eIF4AI in an in vitro-reconstituted 40S ribosome binding assay. Instead, eIF4AIII inhibits translation in a reticulocyte lysate system. In addition, whereas eIF4AI binds independently to the middle and carboxy-terminal fragments of eIF4G, eIF4AIII binds to the middle fragment only. These functional differences between eIF4AI and eIF4AIII suggest that eIF4AIII might play an inhibitory role in translation under physiological conditions.     

The RNA Helicase eIF4A Is Required for Sapovirus Translation

The eukaryotic initiation factor 4A (eIF4A) is a DEAD box helicase that unwinds RNA structure in the 5′ untranslated region (UTR) of mRNAs. Here, we investigated the role of eIF4A in porcine sapovirus VPg-dependent translation. Using inhibitors and dominant-negative mutants, we found that eIF4A is required for viral translation and infectivity, suggesting that despite the presence of a very short 5′ UTR, eIF4A is required to unwind RNA structure in the sapovirus genome to facilitate virus translation.     

A Eukaryotic Translation Initiation Factor 4E-Binding Protein Promotes mRNA Decapping and Is Required for PUF Repression

PUF proteins are eukaryotic RNA-binding proteins that repress specific mRNAs. The mechanisms and corepressors involved in PUF repression remain to be fully identified. Here, we investigated the mode of repression by Saccharomyces cerevisiae Puf5p and Puf4p and found that Puf5p specifically requires Eap1p to repress mRNAs, whereas Puf4p does not. Surprisingly, we observed that Eap1p, which is a member of the eukaryotic translation initiation factor 4E (eIF4E)-binding protein (4E-BP) class of translational inhibitors, does not inhibit the efficient polyribosome association of a Puf5p target mRNA. Rather, we found that Eap1p accelerates mRNA degradation by promoting decapping, and the ability of Eap1p to interact with eIF4E facilitates this activity. Deletion of EAP1 dramatically reduces decapping, resulting in accumulation of deadenylated, capped mRNA. In support of this phenotype, Eap1p associates both with Puf5p and the Dhh1p decapping factor. Furthermore, recruitment of Eap1p to downregulated mRNA is mediated by Puf5p. On the basis of these results, we propose that Puf5p promotes decapping by recruiting Eap1p and associated decapping factors to mRNAs. The implication of these findings is that a 4E-BP can repress protein expression by promoting specific mRNA degradation steps in addition to or in lieu of inhibiting translation initiation.     

Eukaryotic Translation Initiation Factor 4E-Dependent Translation Is Not Essential for Survival of Starved Yeast Cells

The eukaryotic translation initiation factor 4E (eIF4E) interacts with the mRNA 5' cap structure (m(7)GpppX) and is essential for the appropriate translation of the vast majority of eukaryotic mRNAs. Most studies of the yeast Saccharomyces cerevisiae CDC33 gene product, eIF4E, have been carried out with logarithmically growing cells, and little is known about its role in starved, nonproliferating cells that enter the stationary phase (SP). It has previously been found that the rate of translation in SP cells is more than 2 orders of magnitude lower than it is in dividing yeast cells. Here we show that this low rate of translation is essential for maintaining the viability of starved yeast cells that enter SP. Specifically, starved cells whose eIF4A is inactive or treated with cycloheximide rapidly lose viability. Moreover, after heat inactivation of the cdc33 temperature-sensitive product, the synthesis of most proteins is abolished and only a small group of proteins is still produced. Unexpectedly, starved cdc33 mutant cells whose eIF4E is inactive and which therefore fail to synthesize the bulk of their proteins remain viable for long periods of time, indistinguishable from their isogenic wild-type counterparts. Taken together, our results indicate that eIF4E-independent translation is necessary and sufficient for survival of yeast cells during long periods of starvation.     

Requirement of RNA Binding of Mammalian Eukaryotic Translation Initiation Factor 4GI (eIF4GI) for Efficient Interaction of eIF4E with the mRNA Cap

Eukaryotic mRNAs possess a 5′-terminal cap structure (cap), m⁷GpppN, which facilitates ribosome binding. The cap is bound by eukaryotic translation initiation factor 4F (eIF4F), which is composed of eIF4E, eIF4G, and eIF4A. eIF4E is the cap-binding subunit, eIF4A is an RNA helicase, and eIF4G is a scaffolding protein that bridges between the mRNA and ribosome. eIF4G contains an RNA-binding domain, which was suggested to stimulate eIF4E interaction with the cap in mammals. In Saccharomyces cerevisiae, however, such an effect was not observed. Here, we used recombinant proteins to reconstitute the cap binding of the mammalian eIF4E-eIF4GI complex to investigate the importance of the RNA-binding region of eIF4GI for cap interaction with eIF4E. We demonstrate that chemical cross-linking of eIF4E to the cap structure is dramatically enhanced by eIF4GI fragments possessing RNA-binding activity. Furthermore, the fusion of RNA recognition motif 1 (RRM1) of the La autoantigen to the N terminus of eIF4GI confers enhanced association between the cap structure and eIF4E. These results demonstrate that eIF4GI serves to anchor eIF4E to the mRNA and enhance its interaction with the cap structure.The cap structure, m⁷GpppN, is present at the 5′ terminus of all nuclear transcribed eukaryotic mRNAs. Cap-dependent binding of the ribosome to mRNA is mediated by the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E), which forms a complex termed eIF4F together with eIF4G and eIF4A. Mammalian eIF4G, which has two isoforms, eIF4GI and eIF4GII, is a modular, multifunctional protein that binds to poly(A)-binding protein (PABP) (14) and eIF4E (18, 20) via the N-terminal third region. Mammalian eIF4G binds to eIF4A and eIF3 (15) via the middle third region and to eIF4A and Mnk protein kinase at the C-terminal region. eIF4GI also possesses an RNA-binding sequence (2, 9, 33) in the middle region. There are two RNA-binding sites on eIF4GI; one is located amino terminal to the first HEAT domain, and the other is located within the first HEAT domain (23). Mammalian and Saccharomyces cerevisiae eIF4E are similar in size (24 kDa), but mammalian eIF4GI (220 kDa) is larger than its yeast counterpart (150 kDa), as the latter lacks a C-terminal domain corresponding to mammalian eIF4GI (38).The affinity of eIF4E for the cap structure has been a matter of dispute for some time. The earlier works of Carberry et al. (4) and Ueda et al. (39) estimated the equilibrium dissociation constant (K_d) of the eIF4E-cap complex by fluorescence titration to be 2 × 10⁻⁶ to 5 × 10⁻⁶ M depending on the nature of the cap analog. Later on, development of a new methodology for the fluorescence titration experiments yielded K_d values of 10⁻⁷ to 10⁻⁸ (29, 41). The source of the difference with the previous reports was thoroughly analyzed (29, 30). The interaction between the cap structure and eIF4E is dramatically enhanced by eIF4GI. This was first reported by showing that cross-linking of mammalian eIF4E to the cap structure is more efficient when it is a subunit of the eIF4F complex (19) or when it is complexed to eIF4GI (11). A similar enhancement of the binding of eIF4E to the cap structure was observed in yeast (40). However, two very different mechanisms were proposed to explain these observations. For the mammalian system, it was postulated that the middle segment of eIF4GI, which binds RNA, stabilizes the eIF4E interaction with the cap structure (11). This model was based primarily on the finding that in poliovirus-infected cells, eIF4GI is cleaved between its N-terminal third and the middle third, and consequently, eIF4E remains attached to the N-terminal eIF4GI fragment lacking the RNA-binding region. Under these conditions, cross-linking of eIF4E to the cap structure was poor (19, 31). In contrast, in yeast, a strong interaction between the cap structure and eIF4E was achieved using an eIF4G fragment containing the eIF4E-binding site that lacks the RNA-binding region (34, 40). Also, the yeast eIF4G fragment from amino acids 393 to 490 (fragment 393-490), which does not contain the RNA-binding site, forms a right-handed helical ring that wraps around the N terminus of eIF4E. This conformational change was suggested in turn to engender an allosteric enhancement of the association of eIF4E with the cap structure (10). Such an interaction between mammalian eIF4GI and eIF4E has not been reported.To understand the mechanism by which eIF4GI stimulates the interaction of eIF4E with the cap structure in mammals, we reconstituted the eIF4E-cap recognition activity in vitro with purified eIF4E and eIF4GI recombinant proteins. Using a chemical cross-linking assay, we demonstrate that only mammalian eIF4GI fragments possessing RNA-binding activity enhance the cross-linking of eIF4E to the cap structure. Our data provide new insight into the mechanism of cap recognition by the eIF4E-eIF4GI complex.     

The Expression Alteration of BC1 RNA and its Interaction with Eukaryotic Translation Initiation Factor eIF4A Post-Status Epilepticus

Abnormal dendritic sprouting and synaptic remodelling are important pathological features of temporal lobe epilepsy. BC1 RNA is a translation repressor involved in the regulation of the dendritic protein synthesis and mRNA transport, which is essential for dendritic development and plasticity. The expression alteration of BC1 RNA in the pilocarpine induced epilepsy model remains unknown. It is unclear if the interactions between BC1 RNA and eukaryotic initiation factor 4A (eIF4A) exists in this model. The purpose of this study was to investigate the expression changes of BC1 RNA and its interactions with eIF4A post-status epilepticus (SE). Chloride lithium and pilocarpine were used to induce the SE rat model. Either a whole brain or hippocampus tissues were collected at different time points after SE. The expression patterns of BC1 was detected by qPCR and in situ hybridization. The levels of eIF4AI/II protein expression were analyzed via western blotting and immunohistochemistry. The BC1 RNA-eIF4AI/II interaction was determined by electrophoretic mobility shift assay (EMSA). We found that the BC1 RNA levels decreased in hippocampus 3d, 1w and 2w post-SE before the levels recovered. The eIF4AI/II began to rise 3d post-SE and reached the maximum level 1w post-SE. After 1w post-SE the levels decreased in the hippocampal CA1, CA3 and DG subregions. EMSA analysis showed that BC1 RNA specifically interacted with the eIF4AI/II. The BC1 RNA-eIF4AI/II complex reduced to the lowest level 1w post-SE. Our results suggested that BC1 has a negative regulatory correlation with eIF4AI/II, where BC1 RNA could be involved in epileptogenesis by regulating dendritic protein synthesis.     

Eukaryotic Translation Initiation Factor 4G Is Targeted for Proteolytic Cleavage by Caspase 3 during Inhibition of Translation in Apoptotic Cells

Although much is known about the multiple mechanisms which induce apoptosis, comparatively little is understood concerning the execution phase of apoptosis and the mechanism(s) of cell killing. Several reports have demonstrated that cellular translation is shut off during apoptosis; however, details of the mechanism of translation inhibition are lacking. Translation initiation factor 4G (eIF4G) is a crucial protein required for binding cellular mRNA to ribosomes and is known to be cleaved as the central part of the mechanism of host translation shutoff exerted by several animal viruses. Treatment of HeLa cells with the apoptosis inducers cisplatin and etoposide resulted in cleavage of eIF4G, and the extent of its cleavage correlated with the onset and extent of observed inhibition of cellular translation. The eIF4G-specific cleavage activity could be measured in cell lysates in vitro and was inhibited by the caspase inhibitor Ac-DEVD-CHO at nanomolar concentrations. A combination of in vivo and in vitro inhibitor studies suggest the involvement of one or more caspases in the activation and execution of eIF4G cleavage. Furthermore recombinant human caspase 3 was expressed in bacteria, and when incubated with HeLa cell lysates, was shown to produce the same eIF4G cleavage products as those observed in apoptotic cells. In addition, purified caspase 3 caused cleavage of purified eIF4G, demonstrating that eIF4G could serve as a substrate for caspase 3. Taken together, these data suggest that cellular translation is specifically inhibited during apoptosis by a mechanism involving cleavage of eIF4G, an event dependent on caspase activity.     

Spectrin Domain of Eukaryotic Initiation Factor 3a Is the Docking Site for Formation of the a:b:i:g Subcomplex

eIF3a (eukaryotic translation initiation factor 3a), one of the core subunits of the eIF3 complex, has been implicated in regulating translation of different mRNAs and in tumorigenesis. A subcomplex consisting of eIF3a, eIF3b, eIF3g, and eIF3i (eIF3(a:b:i:g)) has also been identified. However, how eIF3a participates in translational regulation and in formation of the eIF3(a:b:i:g) subcomplex remain to be solved. In this study, we used the tandem affinity purification approach in combination with tandem MS/MS and identified the spectrin domain of eIF3a as the docking site for the formation of eIF3(a:b:i:g) subcomplex. Although eIF3b and eIF3i bind concurrently to the spectrin domain of eIF3a within ∼10–15 amino acids apart, eIF3g binds to eIF3a indirectly via binding to the carboxyl-terminal domain of eIF3b. The binding of eIF3b to the spectrin domain of eIF3a occurs in its RNA recognition motif domain where eIF3j also binds in a mutually exclusive manner. Together, we conclude that the spectrin domain of eIF3a is responsible for the formation of eIF3(a:b:i:g) subcomplex and, because of mutually exclusive nature of bindings of eIF3a and eIF3j to eIF3b, different subcomplexes of eIF3 likely exist and may perform noncanonical functions in translational regulation.     

The m Subunit of Murine Translation Initiation Factor eIF3 Maintains the Integrity of the eIF3 Complex and Is Required for Embryonic Development,Homeostasis, and Organ Size Control

Mammalian eIF3 is composed of 13 subunits and is the largest eukaryotic initiation factor. eIF3 plays a key role in protein biosynthesis. However, it is not fully understood how different subunits contribute to the structural integrity and function of the eIF3 complex. Whether eIF3 is essential for embryonic development and homeostasis is also not known. Here, we show that eIF3m null embryos are lethal at the peri-implantation stage. Compound heterozygotes (eIF3m^flox/−) or FABP4-Cre-mediated conditional knock-out mice are lethal at mid-gestation stages. Although the heterozygotes are viable, they show markedly reduced organ size and diminished body weight. Acute ablation of eIF3m in adult mouse liver leads to rapidly decreased body weight and death within 2 weeks; these effects are correlated with a severe decline of protein biogenesis in the liver. Protein analyses reveal that eIF3m deficiency significantly impairs the integrity of the eIF3 complex due to down-regulation of multiple other subunits. Two of the subunits, eIF3f and eIF3h, are stabilized by eIF3m through subcomplex formation. Therefore, eIF3m is required for the structural integrity and translation initiation function of eIF3. Furthermore, not only is eIF3m an essential gene, but its expression level is also important for mouse embryonic development and the control of organ size.     

Deoxyhypusine Modification of Eukaryotic Translation Initiation Factor 5A (eIF5A) Is Essential for Trypanosoma brucei Growth and for Expression of Polyprolyl-containing Proteins

The eukaryotic protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis. Polyamine biosynthesis is essential in T. brucei, and the polyamine spermidine is required for synthesis of a novel cofactor called trypanothione and for deoxyhypusine modification of eukaryotic translation initiation factor 5A (eIF5A). eIF5A promotes translation of proteins containing polyprolyl tracts in mammals and yeast. To evaluate the function of eIF5A in T. brucei, we used RNA interference (RNAi) to knock down eIF5A levels and found that it is essential for T. brucei growth. The RNAi-induced growth defect was complemented by expression of wild-type human eIF5A but not by a Lys-50 mutant that blocks modification by deoxyhypusine. Bioinformatics analysis showed that 15% of the T. brucei proteome contains 3 or more consecutive prolines and that actin-related proteins and cysteine proteases were highly enriched in the group. Steady-state protein levels of representative proteins containing 9 consecutive prolines that are involved in actin assembly (formin and CAP/Srv2p) were significantly reduced by knockdown of eIF5A. Several T. brucei polyprolyl proteins are involved in flagellar assembly. Knockdown of TbeIF5A led to abnormal cell morphologies and detached flagella, suggesting that eIF5A is important for translation of proteins needed for these processes. Potential specialized functions for eIF5A in T. brucei in translation of variable surface glycoproteins were also uncovered. Inhibitors of deoxyhypusination would be expected to cause a pleomorphic effect on multiple cell processes, suggesting that deoxyhypusine/hypusine biosynthesis could be a promising drug target in not just T. brucei but in other eukaryotic pathogens.     

Eukaryotic Release Factor 3 Is Required for Multiple Turnovers of Peptide Release Catalysis by Eukaryotic Release Factor 1

Eukaryotic peptide release factor 3 (eRF3) is a conserved, essential gene in eukaryotes implicated in translation termination. We have systematically measured the contribution of eRF3 to the rates of peptide release with both saturating and limiting levels of eukaryotic release factor 1 (eRF1). Although eRF3 modestly stimulates the absolute rate of peptide release (∼5-fold), it strongly increases the rate of peptide release when eRF1 is limiting (>20-fold). This effect was generalizable across all stop codons and in a variety of contexts. Further investigation revealed that eRF1 remains associated with ribosomal complexes after peptide release and subunit dissociation and that eRF3 promotes the dissociation of eRF1 from these post-termination complexes. These data are consistent with models where eRF3 principally affects binding interactions between eRF1 and the ribosome, either prior to or subsequent to peptide release. A role for eRF3 as an escort for eRF1 into its fully accommodated state is easily reconciled with its close sequence similarity to the translational GTPase EFTu.     

The Eukaryotic Initiation Factor (eIF) 4G HEAT Domain Promotes Translation Re-initiation in Yeast Both Dependent on and Independent of eIF4A mRNA Helicase

Translation re-initiation provides the molecular basis for translational control of mammalian ATF4 and yeast GCN4 mediated by short upstream open reading (uORFs) in response to eIF2 phosphorylation. eIF4G is the major adaptor subunit of eIF4F that binds the cap-binding subunit eIF4E and the mRNA helicase eIF4A and is also required for re-initiation in mammals. Here we show that the yeast eIF4G2 mutations altering eIF4E- and eIF4A-binding sites increase re-initiation at GCN4 and impair recognition of the start codons of uORF1 or uORF4 located after uORF1. The increase in re-initiation at GCN4 was partially suppressed by increasing the distance between uORF1 and GCN4, suggesting that the mutations decrease the migration rate of the scanning ribosome in the GCN4 leader. Interestingly, eIF4E overexpression suppressed both the phenotypes caused by the mutation altering eIF4E-binding site. Thus, eIF4F is required for accurate AUG selection and re-initiation also in yeast, and the eIF4G interaction with the mRNA-cap appears to promote eIF4F re-acquisition by the re-initiating 40 S subunit. However, eIF4A overexpression suppressed the impaired AUG recognition but not the increase in re-initiation caused by the mutations altering eIF4A-binding site. These results not only provide evidence that mRNA unwinding by eIF4A stimulates start codon recognition, but also suggest that the eIF4A-binding site on eIF4G made of the HEAT domain stimulates the ribosomal scanning independent of eIF4A. Based on the RNA-binding activities identified within the unstructured segments flanking the eIF4G2 HEAT domain, we discuss the role of the HEAT domain in scanning beyond loading eIF4A onto the pre-initiation complex.     

A Conserved Interaction between a C-Terminal Motif in Norovirus VPg and the HEAT-1 Domain of eIF4G Is Essential for Translation Initiation

Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5´ m⁷G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5´-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652–1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit in vitro translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy.     

Human Eukaryotic Initiation Factor 4G (eIF4G) Protein Binds to eIF3c, -d,and -e to Promote mRNA Recruitment to the Ribosome

Recruitment of mRNA to the 40S ribosomal subunit requires the coordinated interaction of a large number of translation initiation factors. In mammals, the direct interaction between eukaryotic initiation factor 4G (eIF4G) and eIF3 is thought to act as the molecular bridge between the mRNA cap-binding complex and the 40S subunit. A discrete ∼90 amino acid domain in eIF4G is responsible for binding to eIF3, but the identity of the eIF3 subunit(s) involved is less clear. The eIF3e subunit has been shown to directly bind eIF4G, but the potential role of other eIF3 subunits in stabilizing this interaction has not been investigated. It is also not clear if the eIF4A helicase plays a role in stabilizing the interaction between eIF4G and eIF3. Here, we have used a fluorescence anisotropy assay to demonstrate that eIF4G binds to eIF3 independently of eIF4A binding to the middle region of eIF4G. By using a site-specific cross-linking approach, we unexpectedly show that the eIF4G-binding surface in eIF3 is comprised of the -c, -d and -e subunits. Screening multiple cross-linker positions reveals that eIF4G contains two distinct eIF3-binding subdomains within the previously identified eIF3-binding domain. Finally, by employing an eIF4G-dependent translation assay, we establish that both of these subdomains are required for efficient mRNA recruitment to the ribosome and stimulate translation. Our study reveals unexpected complexity to the eIF3-eIF4G interaction that provides new insight into the regulation of mRNA recruitment to the human ribosome.