Activation of cap-independent translation by variant eukaryotic initiation factor 4G in vivo

Protein synthesis is tightly controlled by assembly of an intricate ribonucleoprotein complex at the m⁷GTP-cap on eukaryotic mRNAs. Ensuing linear scanning of the 5′ untranslated region (UTR) is believed to transfer the preinitiation complex to the initiation codon. Eukaryotic mRNAs are characterized by significant 5′ UTR heterogeneity, raising the possibility of differential control of translation initiation rate at individual mRNAs. Curiously, many mRNAs with unconventional, highly structured 5′ UTRs encode proteins with central biological roles in growth control, metabolism, or stress response. The 5′ UTRs of such mRNAs may influence protein synthesis rate in multiple ways, but most significantly they have been implicated in mediating alternative means of translation initiation. Cap-independent initiation bypasses strict control over the formation of initiation intermediates at the m⁷GTP cap. However, the molecular mechanisms that favor alternative means of ribosome recruitment are not understood. Here we provide evidence that eukaryotic initiation factor (eIF) 4G controls cap-independent translation initiation at the c-myc and vascular endothelial growth factor (VEGF) 5′ UTRs in vivo. Cap-independent translation was investigated in tetracycline-inducible cell lines expressing either full-length eIF4G or a C-terminal fragment (Ct) lacking interaction with eIF4E and poly(A) binding protein. Expression of Ct, but not intact eIF4G, potently stimulated cap-independent initiation at the c-myc/VEGF 5′ UTRs. In vitro RNA-binding assays suggest that stimulation of cap-independent translation initiation by Ct is due to direct association with the c-myc/VEGF 5′ UTR, enabling 43S preinitiation complex recruitment. Our work demonstrates that variant translation initiation factors enable unconventional translation initiation at mRNA subsets with distinct structural features.     

Compartmentalisation and localisation of the translation initiation factor (eIF) 4F complex in normally growing fibroblasts

Previous observations of association of mRNAs and ribosomes with subcellular structures highlight the importance of localised translation. However, little is known regarding associations between eukaryotic translation initiation factors and cellular structures within the cytoplasm of normally growing cells. We have used detergent-based cellular fractionation coupled with immunofluorescence microscopy to investigate the subcellular localisation in NIH3T3 fibroblasts of the initiation factors involved in recruitment of mRNA for translation, focussing on eIF4E, the mRNA cap-binding protein, the scaffold protein eIF4GI and poly(A) binding protein (PABP). We find that these proteins exist mainly in a soluble cytosolic pool, with only a subfraction tightly associated with cellular structures. However, this "associated" fraction was enriched in active "eIF4F" complexes (eIF4E.eIF4G.eIF4A.PABP). Immunofluorescence analysis reveals both a diffuse and a perinuclear distribution of eIF4G, with the perinuclear staining pattern similar to that of the endoplasmic reticulum. eIF4E also shows both a diffuse staining pattern and a tighter perinuclear stain, partly coincident with vimentin intermediate filaments. All three proteins localise to the lamellipodia of migrating cells in close proximity to ribosomes, microtubules, microfilaments and focal adhesions, with eIF4G and eIF4E at the periphery showing a similar staining pattern to the focal adhesion protein vinculin.     

Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: A reappraisal of translation initiation during mitosis

Translation mechanisms at different stages of the cell cycle have been studied for many years, resulting in the dogma that translation rates are slowed during mitosis, with cap-independent translation mechanisms favored to give expression of key regulatory proteins. However, such cell culture studies involve synchronization using harsh methods, which may in themselves stress cells and affect protein synthesis rates. One such commonly used chemical is the microtubule de-polymerization agent, nocodazole, which arrests cells in mitosis and has been used to demonstrate that translation rates are strongly reduced (down to 30% of that of asynchronous cells). Using synchronized HeLa cells released from a double thymidine block (G₁/S boundary) or the Cdk1 inhibitor, RO3306 (G₂/M boundary), we have systematically re-addressed this dogma. Using FACS analysis and pulse labeling of proteins with labeled methionine, we now show that translation rates do not slow as cells enter mitosis. This study is complemented by studies employing confocal microscopy, which show enrichment of translation initiation factors at the microtubule organizing centers, mitotic spindle, and midbody structure during the final steps of cytokinesis, suggesting that translation is maintained during mitosis. Furthermore, we show that inhibition of translation in response to extended times of exposure to nocodazole reflects increased eIF2α phosphorylation, disaggregation of polysomes, and hyperphosphorylation of selected initiation factors, including novel Cdk1-dependent N-terminal phosphorylation of eIF4GII. Our work suggests that effects on translation in nocodazole-arrested cells might be related to those of the treatment used to synchronize cells rather than cell cycle status.     

Second-generation derivatives of the eukaryotic translation initiation inhibitor pateamine A targeting eIF4A as potential anticancer agents

A series of pateamine A (1) derivatives were synthesized for structure/activity relationship (SAR) studies and a selection of previous generation analogs were re-evaluated based on current information regarding the mechanism of action of these translation inhibitors. Structural modifications in the new generation of derivatives focused on alterations to the C19–C22 Z,E-diene and the trienyl side chain of the previously described simplified, des-methyl, des-amino pateamine A (DMDAPatA, 2). Derivatives were tested for anti-proliferative activity in cell culture and for inhibition of mammalian cap-dependent translation in vitro. Activity was highly dependent on the rigidity and conformation of the macrolide and the functionality of the side chain. The only well tolerated substitutions were replacement of the N,N-dimethyl amino group found on the side chain of 2 with other tertiary amine groups. SAR reported here suggests that this site may be modified in future studies to improve serum stability, cell-type specificity, and/or specificity towards rapidly proliferating cells.     

Mutations in the eIF(iso)4G translation initiation factor confer high resistance of rice to Rice yellow mottle virus

We report here evidence of the role that the isoform of the eukaryotic translation initiation factor 4G (eIF(iso)4G) plays in naturally occurring resistance in plant/virus interactions. A genetic and physical mapping approach was developed to isolate the Rymv1 locus controlling the high recessive resistance to Rice yellow mottle virus (RYMV) in the rice (Oryza sativa) variety Gigante. The locus was mapped to a 160-kb interval containing a gene from the eIF(iso)4G family. The stable transformation of a resistant line with the cDNA of this gene, derived from a susceptible variety, resulted in the loss of resistance in transgenic plants. The allelic variability of this gene was analysed in three resistant and 17 susceptible varieties from different cultivated rice species or subspecies. Compared with susceptible varieties, resistant varieties present specific alleles, characterized by either amino acid substitutions or short amino-acid deletions in the middle domain of the protein. The structure of this domain was modelled and showed that the substitutions were clustered on a small surface patch. This suggests that this domain may be involved in an interaction with the virus.     

Plant cap-binding complexes eukaryotic initiation factors eIF4F and eIFISO4F: molecular specificity of subunit binding

The initiation of translation in eukaryotes requires a suite of eIFs that include the cap-binding complex, eIF4F. eIF4F is comprised of the subunits eIF4G and eIF4E and often the helicase, eIF4A. The eIF4G subunit serves as an assembly point for other initiation factors, whereas eIF4E binds to the 7-methyl guanosine cap of mRNA. Plants have an isozyme form of eIF4F (eIFiso4F) with comparable subunits, eIFiso4E and eIFiso4G. Plant eIF4A is very loosely associated with the plant cap-binding complexes. The specificity of interaction of the individual subunits of the two complexes was previously unknown. To address this issue, mixed complexes (eIF4E-eIFiso4G or eIFiso4E-eIF4G) were expressed and purified from Escherichia coli for biochemical analysis. The activity of the mixed complexes in in vitro translation assays correlated with the large subunit of the respective correct complex. These results suggest that the eIF4G or eIFiso4G subunits influence translational efficiency more than the cap-binding subunits. The translation assays also showed varying responses of the mRNA templates to eIF4F or eIFiso4F, suggesting that some level of mRNA discrimination is possible. The dissociation constants for the correct complexes have K(D) values in the subnanomolar range, whereas the mixed complexes were found to have K(D) values in the ～10 nm range. Displacement assays showed that the correct binding partner readily displaces the incorrect binding partner in a manner consistent with the difference in K(D) values. These results show molecular specificity for the formation of plant eIF4F and eIFiso4F complexes and suggest a role in mRNA discrimination during initiation of translation.     

Molecular dissection of the eukaryotic initiation factor 4E (eIF4E) export‐competent RNP

The eukaryotic translation initiation factor 4E (eIF4E) controls gene expression through its effects on mRNA export and cap‐dependent translation, both of which contribute to its oncogenic potential. In contrast to its translation function, the mRNA export function of eIF4E is poorly understood. Using an RNP isolation/mass spectrometry approach, we identified candidate cofactors of eIF4E mRNA export including LRPPRC. This protein associates with mRNAs containing the eIF4E‐sensitivity element (4E‐SE), and its overexpression alters the nuclear export of several eIF4E‐sensitive mRNAs. LRPPRC‐mediated alteration of eIF4E's mRNA export function requires the integrity of its eIF4E‐binding site and it coincides with the subcellular re‐distribution of eIF4E. The eIF4E export RNP is distinct in composition from the bulk mRNA export pathway, in that eIF4E‐ and eIF4E‐sensitive mRNAs do not associate with general mRNA export factors such as TAP/NXF1 or REF/Aly. Our data indicate that mRNA export pathways have evolved for specific mRNAs enabling the differential regulation of biochemical pathways by modulating the expression of groups of genes at the level of their export.     

Synergistic effects of eIF4A and MEK inhibitors on proliferation of NRAS-mutant melanoma cell lines

Activating mutations of the NRAS (neuroblastoma rat sarcoma viral oncogene) protein kinase, present in many cancers, induce a constitutive activation of both the RAS-RAF-MEK-ERK mitogen-activated protein kinase (MAPK) signal transduction pathway and the PI(3)K-AKT-mTOR, pathway. This in turn regulates the formation of the eIF4F eukaryotic translation initiation complex, comprising the eIF4E cap-binding protein, the eIF4G scaffolding protein and the eIF4A RNA helicase, which binds to the 7-methylguanylate cap (m(7)G) at the 5′ end of messenger RNAs. Small molecules targeting MEK (MEKi: MEK inhibitors) have demonstrated activity in NRAS-mutant cell lines and tumors, but resistance sets in most cases within months of treatment. Using proximity ligation assays, that allows visualization of the binding of eIF4E to the scaffold protein eIF4G, generating the active eIF4F complex, we have found that resistance to MEKi is associated with the persistent formation of the eIF4F complex in MEKi-treated NRAS-mutant cell lines. Furthermore, inhibiting the eIF4A component of the eIF4F complex, with a small molecule of the flavagline/rocaglate family, synergizes with inhibiting MEK to kill NRAS-mutant cancer cell lines.     

Communication between eukaryotic translation initiation factors 5 and 1A within the ribosomal pre-initiation complex plays a role in start site selection

During eukaryotic translation initiation, the 43 S ribosomal pre-initiation complex scans the mRNA in search of an AUG codon at which to begin translation. Start codon recognition halts scanning and triggers a number of events that commit the complex to beginning translation at that point on the mRNA. Previous studies in vitro and in vivo have indicated that eukaryotic initiation factors (eIFs) 1, 2 and 5 play key roles in these events. In addition, it was reported recently that the C-terminal domain of eIF1A is involved in maintaining the fidelity of start codon recognition. The molecular mechanisms by which these factors work together to ensure fidelity of start site selection remain poorly understood. Here, we report the quantitative characterization of energetic interactions between eIF1A, eIF5 and the AUG codon in an in vitro reconstituted yeast translation initiation system. Our results show that recognition of an AUG codon by the 43 S complex triggers an interaction between eIF5 and eIF1A, resulting in a shift in the equilibrium between two states of the pre-initiation complex. This AUG-dependent change may be a reorganization from a scanning-competent state to a scanning-incompetent state. Mutations in both eIF1A and eIF5 that increase initiation at non-AUG codons in vivo weaken the interaction between the two factors upon AUG recognition, while specifically strengthening it in response to a UUG codon. These data suggest strongly that the interaction between eIF1A and eIF5 is involved in maintaining the fidelity of start codon recognition in vivo.     

Translation of mRNAs from Vesicular Stomatitis Virus and Vaccinia Virus Is Differentially Blocked in Cells with Depletion of eIF4GI and/or eIF4GII

Cytolytic viruses abrogate host protein synthesis to maximize the translation of their own mRNAs. In this study, we analyzed the eukaryotic initiation factor (eIF) 4G requirement for translation of vesicular stomatitis virus (VSV) and vaccinia virus (VV) mRNAs in HeLa cells using two different strategies: eIF4G depletion by small interfering RNAs or cleavage of eIF4G by expression of poliovirus 2A protease. Depletion of eIF4GI or eIF4GII moderately inhibits cellular protein synthesis, whereas silencing of both factors has only a slightly higher effect. Under these conditions, the extent of VSV protein synthesis is similar to that of nondepleted control cells, whereas VV expression is substantially reduced. Similar results were obtained when eIF4E was depleted. On the other hand, eIF4G cleavage by poliovirus 2A protease strongly inhibits translation of VV protein expression, whereas translation directed by VSV mRNAs is not abrogated, even though VSV mRNAs are capped. Therefore, the requirement for eIF4F activity is different for VV and VSV, suggesting that the molecular mechanism by which their mRNAs initiate their translation is also different. Consistent with these findings, eIF4GI does not colocalize with ribosomes in VSV-infected cells, while eIF2α locates at perinuclear sites coincident with ribosomes.     

Crystal structure of the C-terminal domain of the ϵ subunit of human translation initiation factor eIF2B

Eukaryotic translation initiation factor eIF2B, the guanine nucleotide exchange factor (GEF) for eIF2, catalyzes conversion of eIF2·GDP to eIF2·GTP. The eIF2B is composed of five subunits, α, β, γ, δ and ε, within which the ε subunit is responsible for catalyzing the guanine exchange reaction. Here we present the crystal structure of the C-terminal domain of human eIF2Bε (eIF2Bε-CTD) at 2.0-Å resolution. The structure resembles a HEAT motif and three charge-rich areas on its surface can be identified. When compared to yeast eIF2Bε-CTD, one area involves highly conserved AA boxes while the other two are only partially conserved. In addition, the previously reported mutations in human eIF2Bε-CTD, which are related to the loss of the GEF activity and human VWM disease, have been discussed. Based on the structure, most of such mutations tend to destabilize the HEAT motif.     

Specific domains in yeast translation initiation factor eIF4G strongly bias RNA unwinding activity of the eIF4F complex toward duplexes with 5'-overhangs

During eukaryotic translation initiation, the 43 S ribosomal pre-initiation complex is recruited to the 5'-end of an mRNA through its interaction with the 7-methylguanosine cap, and it subsequently scans along the mRNA to locate the start codon. Both mRNA recruitment and scanning require the removal of secondary structure within the mRNA. Eukaryotic translation initiation factor 4A is an essential component of the translational machinery thought to participate in the clearing of secondary structural elements in the 5'-untranslated regions of mRNAs. eIF4A is part of the 5'-7-methylguanosine cap-binding complex, eIF4F, along with eIF4E, the cap-binding protein, and the scaffolding protein eIF4G. Here, we show that Saccharomyces cerevisiae eIF4F has a strong preference for unwinding an RNA duplex with a single-stranded 5'-overhang versus the same duplex with a 3'-overhang or without an overhang. In contrast, eIF4A on its own has little RNA substrate specificity. Using a series of deletion constructs of eIF4G, we demonstrate that its three previously elucidated RNA binding domains work together to provide eIF4F with its 5'-end specificity, both by promoting unwinding of substrates with 5'-overhangs and inhibiting unwinding of substrates with 3'-overhangs. Our data suggest that the RNA binding domains of eIF4G provide the S. cerevisiae eIF4F complex with a second mechanism, in addition to the eIF4E-cap interaction, for directing the binding of pre-initiation complexes to the 5'-ends of mRNAs and for biasing scanning in the 5' to 3' direction.     

eIF4B and eIF4G Jointly Stimulate eIF4A ATPase and Unwinding Activities by Modulation of the eIF4A Conformational Cycle

Eukaryotic translation initiation factor 4A (eIF4A) is a DEAD-box protein that participates in translation initiation. As an ATP-dependent RNA helicase, it is thought to resolve secondary structure elements from the 5′-untranslated region of mRNAs to enable ribosome scanning. The RNA-stimulated ATPase and ATP-dependent helicase activities of eIF4A are enhanced by auxiliary proteins, but the underlying mechanisms are still largely unknown. Here, we have dissected the effect of eIF4B and eIF4G on eIF4A RNA-dependent ATPase- and RNA helicase activities and on eIF4A conformation. We show for the first time that yeast eIF4B, like its mammalian counterpart, can stimulate RNA unwinding by eIF4A, although it does not affect the eIF4A conformation. The eIF4G middle domain enhances this stimulatory effect and promotes the formation of a closed eIF4A conformation in the presence of ATP and RNA. The closed state of eIF4A has been inferred but has not been observed experimentally before. eIF4B and eIF4G jointly stimulate ATP hydrolysis and RNA unwinding by eIF4A and favor the formation of the closed eIF4A conformer. Our results reveal distinct functions of eIF4B and eIF4G in synergistically stimulating the eIF4A helicase activity in the mRNA scanning process.     

带有穿膜肽重组PTD-KLF4的制备及活性鉴定

KLF4是Kruppel样转录因子(kruppel-like factors, KLF)家族中的一员,是维持胚胎干细胞(embryonic stem cells, ESCs)全能性的重要转录因子。蛋白质转导结构域(protein transd uction domain, PTD)能够携带大分子进入细胞。为了获得具有穿膜功能的重组KLF4蛋白,利用原核表达载体PKYB表达KLF4与PTD的融合蛋白PTD-KLF4,用Ni柱纯化融合蛋白并作Western blotting鉴定。利用异硫氰酸荧光素(fluorescein isothiocyanate, FITC)标记PTD-KLF4检测其穿越中国仓鼠卵巢(chinese hamster ovary, CHO)细胞细胞膜的能力;用荧光共振能量转移(fluorescence resonance energy transfer, FRET)检测重组融合蛋白PTD-KLF4结合目的DNA的活性。结果表明:重组PTD-KLF4可以成功进入细胞、定位细胞核内,穿膜效率为(22.29±2.1)%。重组融合蛋白PTD-KLF4引起细胞形态变化,并具有与目标DNA序列特异结合的能力。重组PTD-KLF4的制备为外源蛋白诱导多能干细胞(induced pluripotent stem cells, IPSCs)奠定基础。     

Molecular modeling of the human eukaryotic translation initiation factor 5A (eIF5A) based on spectroscopic and computational analyses

The eukaryotic translation initiation factor 5A (eIF5A) is a protein ubiquitously present in archaea and eukarya, which undergoes a unique two-step post-translational modification called hypusination. Several studies have shown that hypusination is essential for a variety of functional roles for eIF5A, including cell proliferation and synthesis of proteins involved in cell cycle control. Up to now neither a totally selective inhibitor of hypusination nor an inhibitor capable of directly binding to eIF5A has been reported in the literature. The discovery of such an inhibitor might be achieved by computer-aided drug design based on the 3D structure of the human eIF5A. In this study, we present a molecular model for the human eIF5A protein based on the crystal structure of the eIF5A from Leishmania brasiliensis, and compare the modeled conformation of the loop bearing the hypusination site with circular dichroism data obtained with a synthetic peptide of this loop. Furthermore, analysis of amino acid variability between different human eIF5A isoforms revealed peculiar structural characteristics that are of functional relevance.     

Structure and functions of the translation initiation factor eIF4E and its role in cancer development and treatment

In eukaryotic cells, protein synthesis is a complex and multi-step process that has several mechanisms to start the translation including cap-dependent and cap-independent initiation. The translation control of eukaryotic gene expression occurs principally at the initiation step. In this context, it is critical that the eukaryotic translation initiation factor eIF4E bind to the 7-methylguanosine (m7G) cap present at the 5′-UTRs of most eukaryotic mRNAs. Combined with other initiation factors, eIF4E mediates the mRNA recruitment on ribosomes to start the translation. Moreover, the eIF4E nuclear bodies are involved in the export of specific mRNAs from the nucleus to the cytoplasm. In this review, we focus on the eIF4E structure and its physiological functions, and describe the role of eIF4E in cancer development and progression and the current therapeutic strategies to target eIF4E.     

Interactions and subcellular distribution of DNA replication initiation proteins in eukaryotic cells

For initiation of eukaryotic DNA replication the origin recognition complex (ORC) associates with chromatin sites and constitutes a landing pad allowing Cdc6, Cdt1 and MCM proteins to accomplish the pre-replication complex (pre-RC). In S phase, the putative MCM helicase is assumed to move away from the ORC to trigger DNA unwinding. By using the fluorescence-based assays bioluminescence resonance energy transfer (BRET) and bimolecular fluorescence complementation (BiFC) we show in live mammalian cells that one key interaction in pre-RC assembly, the interaction between Orc2 and Orc3, is not restricted to the nucleus but also occurs in the cytoplasm. BRET assays also revealed a direct interaction between Orc2 and nuclear localization signal (NLS)-depleted Orc3. Further, we assessed the subcellular distribution of Orc2 and Orc3 in relation to MCM proteins Mcm3 and Mcm6 as well as to a key protein involved in elongation of DNA replication, proliferating nuclear cell antigen (PCNA). Our findings illustrate the spatial complexity of the elaborated process of DNA replication as well as that the BRET and BiFC techniques are novel tools that could contribute to our understanding of the processes at the very beginning of the duplication of the genome.     

Characterization of plant eukaryotic translation initiation factor 6 (eIF6) genes: The essential role in embryogenesis and their differential expression in Arabidopsis and rice

Eukaryotic translation initiation factor 6 (eIF6) is an essential component of ribosome biogenesis. In our present study, we characterize plant eIF6 genes for the first time. Although a single gene encodes eIF6 in yeast and animals, two genes were found to encode proteins homologous to animal and yeast eIF6 in Arabidopsis and rice, denoted At-eIF6;1 and At-eIF6;2, and Os-eIF6;1 and Os-eIF6;2, respectively. Analysis of the yeast eif6 (tif6) mutant suggested that plant eIF6, at least in the case of At-eIF6;1, can complement the essential function of eIF6 in yeast. Evidence for the essential role of eIF6 in plants was also provided by the embryonic-lethal phenotype of the at-eif6;1 mutant. In contrast, At-eIF6;2 appears not to be essential due to its very low expression level and the normal growth phenotype of the eif6;2 mutants. Consistent with the putative role of plant eIF6 in ribosome biogenesis, At-eIF6;1 is predominately expressed in tissues where cell division actively proceeds under the control of intronic cis-regulatory elements. On the other hand, both Os-eIF6;1 and Os-eIF6;2 are probably active genes because they are expressed at significant expression levels. Interestingly, the supply of ammonium nitrate as a plant nutrient was found to induce specifically the expression of Os-eIF6;2. Our present findings indicate that the eIF6 genes have differently evolved in plant and animal kingdoms and also in distinct plant species.