GRP94 (endoplasmin) co-purifies with and is phosphorylated by Golgi apparatus casein kinase

Certain picornaviruses encode proteinases which cleave the translation initiation factor eIF4G, a member of the eIF4F complex which recruits mRNA to the 40S ribosomal subunit during initiation of protein synthesis in eukaryotes. We have compared the efficiency of eIF4G cleavage in rabbit reticulocyte lysates during translation of mRNAs encoding the foot-and-mouth disease virus leader proteinase (L^pro) or the human rhinovirus 2A^pro. Under standard translation conditions, L^pro cleaved 50% of eIF4G within 4 min after initiation of protein synthesis, whereas 2A^pro required 15 min. At these times, the molar ratios of proteinase to eIF4G were 1:130 for L^pro and 1:12 for 2A^pro, indicating a much more efficient in vitro cleavage than previously observed. The molar ratios are similar to those observed during viral infection in vivo.     

The eukaryotic translation initiation factor 4GI is cleaved by different retroviral proteases

The initiation factor eIF4G plays a central role in the regulation of translation. In picornaviruses, as well as in human immunodeficiency virus type 1 (HIV-1), cleavage of eIF4G by the viral protease leads to inhibition of protein synthesis directed by capped cellular mRNAs. In the present work, cleavage of both eIF4GI and eIF4GII has been analyzed by employing the proteases encoded within the genomes of several members of the family Retroviridae, e.g., Moloney murine leukemia virus (MoMLV), mouse mammary tumor virus, human T-cell leukemia virus type 1, HIV-2, and simian immunodeficiency virus. All of the retroviral proteases examined were able to cleave the initiation factor eIF4GI both in intact cells and in cell-free systems, albeit with different efficiencies. The eIF4GI hydrolysis patterns obtained with HIV-1 and HIV-2 proteases were very similar to each other but rather different from those obtained with MoMLV protease. Both eIF4GI and eIF4GII were cleaved very efficiently by the MoMLV protease. However, eIF4GII was a poor substrate for HIV proteases. Proteolytic cleavage of eIF4G led to a profound inhibition of cap-dependent translation, while protein synthesis driven by mRNAs containing internal ribosome entry site elements remained unaffected or was even stimulated in transfected cells.     

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.     

elF4G and its proteolytic cleavage products: effect on initiation of protein synthesis from capped, uncapped, and IRES-containing mRNAs.

Rhinovirus 2A and foot-and-mouth disease virus Lb proteinases stimulate the translation of uncapped messages and those carrying the rhinovirus and enterovirus Internal Ribosome Entry Segments (IRESes) by a mechanism involving the cleavage of host cell proteins. Here, we investigate this mechanism using an artificial dicistronic RNA containing the human rhinovirus IRES as intercistronic spacer. Because both proteinases cleave eukaryotic initiation factor 4G (eIF4G), we examined whether the cleavage products of eIF4G could stimulate uncapped or IRES-driven translation. Addition of intact eIF4F to translation extracts inhibited IRES-driven translation and reduced the translation stimulation observed in reactions pre-treated with Lb proteinase. Prolonged incubation of translation extracts with Lb proteinase removed all endogenous eIF4G and a substantial amount of the primary C- and N-terminal cleavage products. The translation of all mRNAs was reduced in such extracts. Capped mRNA translation was rescued by the addition of intact eIF4F. In contrast, addition of pre-cleaved eIF4F stimulated translation of uncapped or IRES-bearing messages to the levels seen upon proteinase addition. Furthermore, fractions containing the C-terminal, but not N-terminal, cleavage product of eIF4G stimulated translation moderately. These results demonstrate that the Lb and 2A proteinases stimulate translation of uncapped RNAs and those carrying IRESes by the production of cleavage products of eIF4G that enhance translation and by the removal of intact eIF4G that interferes with this stimulation.     

Cap-independent polysomal association of natural mRNAs encoding c-myc, BiP, and eIF4G conferred by internal ribosome entry sites.

Sequence elements that can function as internal ribosome entry sites (IRES) have been identified in 5' noncoding regions of certain uncapped viral and capped cellular mRNA molecules. However, it has remained largely unknown whether IRES elements are functional when located in their natural capped mRNAs. Therefore, the polysomal association and translation of several IRES-containing cellular mRNAs was tested under conditions that severely inhibited cap-dependent translation, that is, after infection with poliovirus. It was found that several known IRES-containing mRNAs, such as BiP and c-myc, were both associated with the translation apparatus and translated in infected cells when cap-dependent translation of most host-cell mRNAs was blocked, indicating that the IRES elements were functional in their natural mRNAs. Curiously, the mRNAs that encode eukaryotic initiation factor 4GI (eIF4GI) and 4GII (eIF4GII), two proteins with high identity and similar functions in the initiation of cap-dependent translation, were both associated with polysomes in infected cells. The 5'-end sequences of eIF4GI mRNA were isolated from a cDNA expression library and shown to function as an internal ribosome entry site when placed into a dicistronic mRNA. These findings suggest that eIF4G proteins can be synthesized at times when 5' cap-dependent mRNA translation is blocked, supporting the notion that eIF4G proteins are needed in both 5' cap-independent and 5' cap-dependent translational initiation mechanisms.     

Cap-independent translation initiation in Xenopus oocytes.

Eukaryotic cellular mRNAs contain a cap at their 5'-ends, but some viral and cellular mRNAs bypass the cap-dependent mechanism of translation initiation in favor of internal entry of ribosomes at specific RNA sequences. Cap-dependent initiation requires intact initiation factor eIF4G (formerly eIF-4gamma, eIF-4Fgamma or p220), whereas internal initiation can proceed with eIF4G cleaved by picornaviral 2A or L proteases. Injection of recombinant coxsackievirus B4 protease 2A into Xenopus oocytes led to complete cleavage of endogenous eIF4G, but protein synthesis decreased by only 35%. Co-injection of edeine reduced synthesis by >90%, indicating that eIF4G-independent synthesis involved ongoing initiation. The spectrum of endogenous proteins synthesized was very similar in the presence or absence of intact eIF4G. Translation of exogenous rabbit globin mRNA, by contrast, was drastically inhibited by eIF4G cleavage. The N-terminal cleavage product of eIF4G (cpN), which binds eIF4E, was completely degraded within 6-12 h, while the C-terminal cleavage product (cpC), which binds to eIF3 and eIF4A, was more stable over the same period. Thus, translation initiation of most endogenous mRNAs inXenopusoocytes requires no eIF4G, or perhaps only cpC, suggesting a cap-independent mechanism.     

Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5′-Terminal Cap and Hairpin

The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5′-ends, we show that an MNK inhibitor, {"type":"entrez-protein","attrs":{"text":"CGP57380","term_id":"877393391","term_text":"CGP57380"}}CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5′-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357–1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m⁷GTP-Sepharose reveals that both CGP and eIF4G(1357–1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5′-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F.     

Equol,an Isoflavone Metabolite,Regulates Cancer Cell Viability and Protein Synthesis Initiation via c-Myc and eIF4G

Epidemiological studies implicate dietary soy isoflavones as breast cancer preventives, especially due to their anti-estrogenic properties. However, soy isoflavones may also have a role in promoting breast cancer, which has yet to be clarified. We previously reported that equol, a metabolite of the soy isoflavone daidzein, may advance breast cancer potential via up-regulation of the eukaryotic initiation factor 4GI (eIF4GI). In estrogen receptor negative (ER−) metastatic breast cancer cells, equol induced elevated levels of eIF4G, which were associated with increased cell viability and the selective translation of mRNAs that use non-canonical means of initiation, including internal ribosome entry site (IRES), ribosome shunting, and eIF4G enhancers. These mRNAs typically code for oncogenic, survival, and cell stress molecules. Among those mRNAs translationally increased by equol was the oncogene and eIF4G enhancer, c-Myc. Here we report that siRNA-mediated knockdown of c-Myc abrogates the increase in cancer cell viability and mammosphere formation by equol, and results in a significant down-regulation of eIF4GI (the major eIF4G isoform), as well as reduces levels of some, but not all, proteins encoded by mRNAs that are translationally stimulated by equol treatment. Knockdown of eIF4GI also markedly reduces an equol-mediated increase in IRES-dependent mRNA translation and the expression of specific oncogenic proteins. However, eIF4GI knockdown did not reciprocally affect c-Myc levels or cell viability. This study therefore implicates c-Myc as a potential regulator of the cancer-promoting effects of equol via up-regulation of eIF4GI and selective initiation of translation on mRNAs that utilize non-canonical initiation, including certain oncogenes.     

Mextli Is a Novel Eukaryotic Translation Initiation Factor 4E-Binding Protein That Promotes Translation in Drosophila melanogaster

Translation is a fundamental step in gene expression, and translational control is exerted in many developmental processes. Most eukaryotic mRNAs are translated by a cap-dependent mechanism, which requires recognition of the 5′-cap structure of the mRNA by eukaryotic translation initiation factor 4E (eIF4E). eIF4E activity is controlled by eIF4E-binding proteins (4E-BPs), which by competing with eIF4G for eIF4E binding act as translational repressors. Here, we report the discovery of Mextli (Mxt), a novel Drosophila melanogaster 4E-BP that in sharp contrast to other 4E-BPs, has a modular structure, binds RNA, eIF3, and several eIF4Es, and promotes translation. Mxt is expressed at high levels in ovarian germ line stem cells (GSCs) and early-stage cystocytes, as is eIF4E-1, and we demonstrate the two proteins interact in these cells. Phenotypic analysis of mxt mutants indicates a role for Mxt in germ line stem cell (GSC) maintenance and in early embryogenesis. Our results support the idea that Mxt, like eIF4G, coordinates the assembly of translation initiation complexes, rendering Mxt the first example of evolutionary convergence of eIF4G function.     

The C-terminal domain of eukaryotic protein synthesis initiation factor (eIF) 4G is sufficient to support cap-independent translation in the absence of eIF4E.

The foot and mouth disease virus, a picornavirus, encodes two forms of a cysteine proteinase (leader or L protease) that bisects the EIF4G polypeptide of the initiation factor complex eIF4F into N-terminal (Nt) and C-terminal (Ct) domains. Previously we showed that, although in vitro cleavage of the translation initiation factor, eIF4G, with L protease decreases cap-dependent translation, the cleavage products themselves may directly promote cap-dependent protein synthesis. We now demonstrate that translation of uncapped mRNAs normally exhibits a strong requirement for eIF4F. However, this dependence is abolished when eIF4G is cleaved, with the Ct domain capable of supporting translation in the absence of the Nt domain. In contrast, the efficient translation of the second cistron of bicistronic mRNAs, directed by two distinct Internal Ribosome Entry Segments (IRES), exhibits no requirement for eIF4E but is dependent upon either intact eIF4G or the Ct domain. These results demonstrate that: (i) the apparent requirement for eIF4F for internal initiation on IRES-driven mRNAs can be fulfilled by the Ct proteolytic cleavage product; (ii) when eIF4G is cleaved, the Ct domain can also support cap-independent translation of cellular mRNAs not possessing an IRES element, in the absence of eIF4E; and (iii) when eIF4G is intact, translation of cellular mRNAs, whether capped or uncapped, is strictly dependent upon eIF4E. These data complement recent work in other laboratories defining the binding sites for other initiation factors on the eIF4G molecule.     

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.     

Protein synthesis initiation factor 4G

eIF4G is a member of the class of translational initiation factors involved in mRNA recruitment to the 43S initiation complex. The proteins from yeast to mammals are present in multiple isoforms of 82-176 kDa. Mammalian eIF4G-1 is synthesized by internal initiation of translation and is specifically degraded by viral and host proteases activated by stress conditions. The role of eIF4G in protein synthesis is inferred from the presence of binding sites for other initiation factors that serve to co-localize the 5'- and 3'-termini of mRNA with RNA-helicase activity and the 40S ribosomal subunit. Growth-regulated mRNAs are preferentially translated under conditions of accentuated eIF4E-eIF4G interaction. Proteolysis of eIF4G or expression of competitor proteins interferes with its binding to either the 5'- or 3'-termini, changing the spectrum of mRNAs translated. Elevated eIF4G levels correlate with malignant cell transformation and diminished eIF4G levels, with nutritional deprivation and anoxia.     

Targeting the eIF4F translation initiation complex for cancer therapy

Abstract In multiple human cancers, the function of the eukaryotic translation initiation factor 4E (eIF4E) is elevated and directly related to disease progression. Overexpression or hyperactivation of eIF4E in experimental models can drive cellular transformation and malignant progression. Elevated eIF4E function triggers enhanced assembly of the eIF4F translation initiation complex and thereby drives cap-dependent translation. Though all capped mRNAs require eIF4F for translation, a pool of mRNAs are exceptionally dependent on elevated eIF4F activity for translation and are thereby selectively and disproportionately affected by altered eIF4F activity. These mRNAs encode proteins that play significant roles in all aspects of malignancy including angiogenesis factors (VEGF, FGF-2), onco-proteins (c-myc, cyclin D1, ODC), pro-survival proteins (survivin, BCL-2) and proteins involved in tumor invasion and metastasis (MMP-9, heparanase). Recent advances in targeting the eIF4F complex have highlighted the role for this complex in tumor cell survival and angiogenesis and have illuminated the enhanced susceptibility of the tumor cells to inhibition of the eIF4F complex. These studies have demonstrated the attractiveness and plausibility of targeting eIF4E and the eIF4F translation initiation complex for cancer therapy and have prompted the advance of the first eIF4E-specific therapy to the clinic.     

Translation of Sindbis virus 26S mRNA does not require intact eukariotic initiation factor 4G

The infection of baby hamster kidney (BHK) cells by Sindbis virus gives rise to a drastic inhibition of cellular translation, while under these conditions the synthesis of viral structural proteins directed by the subgenomic 26S mRNA takes place efficiently. Here, the requirement for intact initiation factor eIF4G for the translation of this subgenomic mRNA has been examined. To this end, SV replicons that contain the protease of human immunodeficiency virus type 1 (HIV-1) or the poliovirus 2A(pro) replacing the sequences of SV glycoproteins have been constructed. BHK cells electroporated with the different RNAs synthesize protein C and the corresponding protease at late times. Notably, the proteolysis of eIF4G by both proteases has little effect on the translation of the 26S mRNA. In addition, recombinant viable SVs were engineered that encode HIV-1 PR or poliovirus 2A protease under the control of a duplicated late promoter. Viral protein synthesis at late times of infection by the recombinant viruses is slightly affected in BHK cells that contain proteolysed eIF4G. The translatability of SV genomic 49S mRNA was assayed in BHK cells infected with a recombinant virus that synthesizes luciferase and transfected with a replicon that expresses poliovirus 2Apro. Under conditions where eIF4G has been hydrolysed significantly the translation of genomic SV RNA was deeply inhibited. These findings indicate a different requirement for intact eIF4G in the translation of genomic and subgenomic SV mRNAs. Finally, the translation of the reporter gene that encodes green fluorescent protein, placed under the control of a second duplicate late promoter, is also resistant to the cleavage of eIF4G. In conclusion, despite the presence of a cap structure in the 5' end of the subgenomic SV mRNA, intact eIF4G is not necessary for its translation.     

Hypoxia-inducible Factor-1α (HIF-1α) Promotes Cap-dependent Translation of Selective mRNAs through Up-regulating Initiation Factor eIF4E1 in Breast Cancer Cells under Hypoxia Conditions

Hypoxia promotes tumor evolution and metastasis, and hypoxia-inducible factor-1α (HIF-1α) is a key regulator of hypoxia-related cellular processes in cancer. The eIF4E translation initiation factors, eIF4E1, eIF4E2, and eIF4E3, are essential for translation initiation. However, whether and how HIF-1α affects cap-dependent translation through eIF4Es in hypoxic cancer cells has been unknown. Here, we report that HIF-1α promoted cap-dependent translation of selective mRNAs through up-regulation of eIF4E1 in hypoxic breast cancer cells. Hypoxia-promoted breast cancer tumorsphere growth was HIF-1α-dependent. We found that eIF4E1, not eIF4E2 or eIF4E3, is the dominant eIF4E family member in breast cancer cells under both normoxia and hypoxia conditions. eIF4E3 expression was largely sequestered in breast cancer cells at normoxia and hypoxia. Hypoxia up-regulated the expression of eIF4E1 and eIF4E2, but only eIF4E1 expression was HIF-1α-dependent. In hypoxic cancer cells, HIF-1α-up-regulated eIF4E1 enhanced cap-dependent translation of a subset of mRNAs encoding proteins important for breast cancer cell mammosphere growth. In searching for correlations, we discovered that human eIF4E1 promoter harbors multiple potential hypoxia response elements. Furthermore, using chromatin immunoprecipitation (ChIP) and luciferase and point mutation assays, we found that HIF-1α utilized hypoxia response elements in the human eIF4E1 proximal promoter region to activate eIF4E1 expression. Our study suggests that HIF-1α promotes cap-dependent translation of selective mRNAs through up-regulating eIF4E1, which contributes to tumorsphere growth of breast cancer cells at hypoxia. The data shown provide new insights into protein synthesis mechanisms in cancer cells at low oxygen levels.     

Specific isoforms of translation initiation factor 4GI show differences in translational activity

The eukaryotic initiation factor (eIF) 4GI gene locus (eIF4GI) contains three identified promoters, generating alternately spliced mRNAs, yielding a total of five eIF4GI protein isoforms. Although eIF4GI plays a critical role in mRNA recruitment to the ribosomes, little is known about the functions of the different isoforms, their partner binding capacities, or the role of the homolog, eIF4GII, in translation initiation. To directly address this, we have used short interfering RNAs (siRNAs) expressed from DNA vectors to silence the expression of eIF4GI in HeLa cells. Here we show that reduced levels of specific mRNA and eIF4GI isoforms in HeLa cells promoted aberrant morphology and a partial inhibition of translation. The latter reflected dephosphorylation of 4E-BP1 and decreased eIF4F complex levels, with no change in eIF2alpha phosphorylation. Expression of siRNA-resistant Myc-tagged eIF4GI isoforms has allowed us to show that the different isoforms exhibit significant differences in their ability to restore translation rates. Here we quantify the efficiency of eIF4GI promoter usage in mammalian cells and demonstrate that even though the longest isoform of eIF4GI (eIF4GIf) was relatively poorly expressed when reintroduced, it was more efficient at promoting the translation of cellular mRNAs than the more highly expressed shorter isoforms used in previous functional studies.     

Tethering of Poly(A)-binding Protein Interferes with Non-translated mRNA Decay from the 5′ End in Yeast

The decay of eukaryotic mRNA is triggered mainly by deadenylation, which leads to decapping and degradation from the 5′ end of an mRNA. Poly(A)-binding protein has been proposed to inhibit the decapping process and to stabilize mRNA by blocking the recruitment of mRNA to the P-bodies where mRNA degradation takes place after stimulation of translation initiation. In contrast, several lines of evidence show that poly(A)-binding protein (Pab1p) has distinct functions in mRNA decay and translation in yeast. To address the translation-independent function of Pab1p in inhibition of decapping, we examined the contribution of Pab1p to the stability of non-translated mRNAs, an AUG codon-less mRNA or an mRNA containing a stable stem-loop structure at the 5′-UTR. Tethering of Pab1p stabilized non-translated mRNAs, and this stabilization did not require either the eIF4G-interacting domain of Pab1p or the Pab1p-interacting domain of eIF4G. In a ski2Δ mutant in which 3′ to 5′ mRNA degradation activity is defective, stabilization of non-translated mRNAs by the tethering of Pab1p lacking an eIF4G-interacting domain (Pab1–34Cp) requires a cap structure but not a poly(A) tail. In wild type cells, stabilization of non-translated mRNA by tethered Pab1–34Cp results in the accumulation of deadenylated mRNA. These results strongly suggest that tethering of Pab1p may inhibit the decapping reaction after deadenylation, independent of translation. We propose that Pab1p inhibits the decapping reaction in a translation-independent manner in vivo.