Proto-oncogenic isoform A2 of eukaryotic translation elongation factor eEF1 is a target of miR-663 and miR-744.

BACKGROUND: Eukaryotic translation elongation factor 1A2 (eEF1A2) is a known proto-oncogene. We proposed that stimulation of the eEF1A2 expression in cancer tissues is caused by the loss of miRNA-mediated control. METHODS: Impact of miRNAs on eEF1A2 at the mRNA and protein levels was examined by qPCR and western blot, respectively. Dual-luciferase assay was applied to examine the influence of miRNAs on 3'-UTR of EEF1A2. To detect miRNA-binding sites, mutations into the 3'-UTR of EEF1A2 mRNA were introduced by the overlap extension PCR. RESULTS: miR-663 and miR-744 inhibited the expression of luciferase gene attached to the 3'-UTR of EEF1A2 up to 20% and 50%, respectively. In MCF7 cells, overexpression of miR-663 and miR-744 reduced the EEF1A2 mRNA level by 30% and 50%. Analogous effects were also observed at the eEF1A2 protein level. In resveratrol-treated MCF7 cells the upregulation of mir-663 and mir-744 was accompanied by downregulation of EEF1A2 mRNA. Both miRNAs were able to inhibit the proliferation of MCF7 cells. CONCLUSION: miR-663 and miR-744 mediate inhibition of the proto-oncogene eEF1A2 expression that results in retardation of the MCF7 cancer cells proliferation. Antitumour effect of resveratrol may include stimulation of the miR-663 and miR-744 expression.

The eEF1 family of mammalian translation elongation factors.

The eEF1 family of mammalian translation elongation factors is comprised of the two variants of eEF1A (eEF1A1 and eEF1A2), and the eEF1B complex. The latter consists of eEF1Bα, eEF1Bß, and eEF1Bγ subunits. The two eEF1A variants have similar translation activity but may differ with respect to their secondary, "moonlighting" functions. This variability is underlined by the difference in the spatial organization of eEF1A1 and eEF1A2, and also possibly by the differences in their post-translational modifications. Here, we review the data on the spatial organization and post-translation modifications of eEF1A1 and eEF1A2, and provide examples of their involvement in various processes in addition to translation. We also describe the structural models of eEF1B subunits, their organization in the subcomplexes, and the trimeric model of the entire eEF1B complex. We discuss the functional consequences of such an assembly into a complex as well as the involvement of individual subunits in non-translational processes.

Purification, crystallization and preliminary X-ray crystallographic analysis of mammalian translation elongation factor eEF1A2.

Translation elongation factor eEF1A2 was purified to homogeneity from rabbit muscle by two consecutive ion-exchange column-chromatography steps and this mammalian eEF1A2 was successfully crystallized for the first time. Protein crystals obtained using ammonium sulfate as precipitant diffracted to 2.5 Å resolution and belonged to space group P6(1)22 or P6(3)22 (unit-cell parameters a = b = 135.4, c = 304.6 Å). A complete native data set was collected to 2.7 Å resolution.

Eukaryotic translation elongation factor 1 alpha: structure, expression, functions, and possible role in aminoacyl-tRNA channeling.

This review offers a comprehensive analysis of eukaryotic translation elongation factor 1 (eEF-1 alpha) in comparison with its bacterial counterpart EF-Tu. Altogether, the data presented indicate some variances in the elongation process in prokaryotes and eukaryotes. The differences may be attributed to translational channeling and compartmentalization of protein synthesis in higher eukaryotic cells. The functional importance of the EF-1 multisubunit complex and expression of its subunits under miscellaneous cellular conditions are reviewed. A number of novel functions of EF-1 alpha, which may contribute to the coordinate regulation of multiple cellular processes including growth, division, and transformation, are characterized.

Chaperone-like activity of mammalian elongation factor eEF1A: renaturation of aminoacyl-tRNA synthetases.

Eukaryotic translational elongation factor eEF1A is known to be responsible for the binding of codon-specific aminoacyl-tRNAs to the ribosome. In this study, we report that in addition to this canonical function, eEF1A is able to promote the renaturation of aminoacyl-tRNA synthetases (ARS) and protect them against denaturation by dilution. The full recovery of the phenylalanyl- (PheRS) and seryl-tRNA synthetase (SerRS) activities was achieved in the presence of 4 microM eEF1A, while bovine serum albumin at similar concentration had no renaturation effect. Remarkably, in vitro renaturation occurs at the molar ratio of eEF1A to ARS equivalent to that found in the cytoplasm of higher eukaryotic cells. The eEF1A.GDP and eEF1A.GTP complexes were shown to be similar in their effect on the phenylalanyl-tRNA synthetase renaturation. Thus, we conclude that the chaperone-like activity of eEF1A might be important for maintaining the enzymes activity in the protein synthesis compartments of mammalian cells.

Rabbit translation elongation factor 1 alpha stimulates the activity of homologous aminoacyl-tRNA synthetase.

Functional and structural sequestration of aminoacyl-tRNA has been recently found in eukaryotic cells and the aminoacyl-tRNA channeling has been suggested [B.S. Negrutskii et al., Proc. Natl. Acad. Sci. 91 (1994) 964-968], but molecular details and mechanism of the process remained unclear. In this paper we have verified a possible interaction between rabbit aminoacyl-tRNA synthetase and homologous translation elongation factor 1 alpha (EF-1 alpha), the proteins which may play a role of sequential components involved in the transfer of the aminoacyl-tRNA along the protein synthetic metabolic chain. The stimulation of the phenylalanyl-tRNA synthetase activity by EF-1 alpha is found. The effect is shown to be specific towards the origin of tRNA and elongation factor molecules. The data obtained favor the direct transfer mechanism of the aminoacyl-tRNA channeling process during eukaryotic protein synthesis.

Evidence for the formation of an unusual ternary complex of rabbit liver EF-1alpha with GDP and deacylated tRNA.

Eukaryotic translation elongation factor 1alpha is known to interact in GTP-bound form with aminoacyl-tRNA promoting its binding to the ribosome. In this paper another ternary complex [EF-1alpha*GDP*deacylated tRNA], never considered in widely accepted elongation schemes, is reported for the first time. The formation of this unusual complex, postulated earlier (FEBS Lett. (1996) 382, 18-20), has been detected by four independent methods. [EF-1alpha*GDP]-interacting sites are located in the acceptor stem, TpsiC stem and TpsiC loop of tRNA(Phe) and tRNA(Leu) molecules. Both tRNA and EF-1alpha are found to undergo certain conformational changes during their interaction. The ability of EF-1alpha to form a complex with deacylated tRNA indicates that the factor may perform an important role in tRNA and aminoacyl-tRNA channeling in higher eukaryotic cells.

[Characteristic features of heat inactivation of leucyl-tRNA- synthetase from the rabbit liver in the presence of various tRNA conformers]. / Osobennosti teplovoi inaktivatsii leitsil-tRNK-sintetazu iz pecheni krolika v prisutstvii razlichnykh konformerov tRNK.

The effect of biologically inactive and active tRNA conformers on heat inactivation of leucyl-tRNA synthetase was investigated. The data presented show that inactive tRNA conformers seem to form complexes with leucyl-tRNA synthetase, but the thermal stability of the enzyme involved in the complex with inactive and active tRNA conformers is rather different.

[Functional compartmentation of the translation apparatus and channeling of tRNA/aminoacyl-tRNA in cells of higher eukaryotes]. / Funktsional'naia kompartmentalizatsiia apparata transliatsii i kanalizovannyi perenos tRNK/aminoatsil-tRNK v kletkakh vysshykh éukariot.

Literature data and authors' results in the field of structure-function organization of the translation apparatus in higher eukaryotes are considered. Proof is presented of the channeling of tRNA/aminoacyl-tRNA in the course of eukaryotic protein synthesis. The concept of the shuttle role of elongation factor eEF1A is grounded; the factor, being in a GTP-bound form, delivers aminoacyl-tRNA to the ribosome and then, in the GDP form after hydrolysis of GTP on the ribosome, forms a complex with the deacylated tRNA and delivers it to the aminoacyl-tRNA synthetase. The notion of translational compartment is defined.

[Influence of various tRNA conformers on mRNA translation in cell-free protein-synthesizing systems]. / Vliianie razlichnykh konformerov tRNK na transliatsiiu mRNK v beskletochnykh beloksinteziruiushchikh sistemakh.

It is found that both globin mRNA translation in the wheat germ system and reticulocyte lysate protein synthesis are inhibited by the excess of exogenous tRNA. The inhibition was more expressed in the presence of inactive tRNA conformers. The data obtained confirm the possibility in principle of inactive tRNA conformers participation in negative regulation of protein synthesis.

[Code correspondence of tRNK Leu 1-6 in the mammary gland of cows]. / Izuchenie kodovogo sootvetstviia tRNK Leu 1-6 molochnoi zhelezy korov.

Preparations of ribosome subunits from the rabbit liver are completely free from the endogenic mRNA admixture and high-active in a system of codon-dependent binding of tRNA. Leucine codons of uridyl-uridyl-adenine (UUA) and uridyl-uridyl-guanine (UUG) are synthesized by the triester method. The correspondence of six leucine isoacceptor tRNA from mammary gland of cows to cytidyl-uridyl-uridine (CUU), UUA and UUG codons is studied. The code response of tRNA is compared with their ability to erroneously translate polyuridylic acid.

[Preparation of monospecific antibodies against isoform 2 of translation elongation factor 1A (eEF1A2)].

Amino acid sequences of eukaryotic translation elongation factor isoform 1 (eEF1A1) and 2 (eEF1A2) were compared and two peptide fragments of eEF1A2 were chosen as linear antigenic determinants for generation of monospecific antipeptide antibodies. Selected peptides were synthesized, conjugated to bovine serum albumin (BSA) and used for mice immunizations. Antibodies, produced against the eEF1A2 fragment 330-343 conjugated to BSA, specifically recognized this isoform in the native and partially denatured states but did not interact with the eEF1A1 isoform. It was shown that these monospecific anti-eEF1A2 antibodies could be employed for eEF1A2 detection both by enzyme-linked immunosorbent assay and by immunoblotting.

The interaction between biologically inactive tRNA conformers and leucyl-tRNA synthetase from rabbit liver.

The interaction between tRNA conformers inactive in aminoacylation and leucyl-tRNA synthetase has been investigated. Heat inactivation of the enzyme in the presence of inactive tRNA conformers is shown to lead to a marked increase of inactivation rate while active tRNA conformers, on the other hand, reveal a protecting effect. To study the properties of the enzyme complexed with different tRNA conformers limited proteolysis has been used. Active tRNA conformers are found to protect leucyl-tRNA synthetase against hydrolysis while inactive ones tend to intensify it. Inactive tRNA conformers are also shown to inhibit the aminoacylation of native tRNA in vitro. On the basis of these data biologically inactive conformers of animal tRNA are assumed to form an unproductive complex with leucyl-tRNA synthetase and the structure of the enzyme involved in such interaction is supposed to be more labile and 'extended' than that in complex with active tRNA conformers.

A sequestered pool of aminoacyl-tRNA in mammalian cells.

We have recently proposed that aminoacyl-tRNA is channeled during protein synthesis in vivo--i.e., it is directly transferred among the components of the protein-synthesizing machinery and does not mix with aminoacyl-tRNA molecules introduced from outside the cell. To understand the structural basis for these functional properties, we have examined the disposition of aminoacyl-tRNA within the cell. To do this we have developed a Chinese hamster ovary (CHO) permeabilized-cell system using the plant glycoside saponin. We show, using a mixture of free 14C-labeled amino acids and 3H-labeled aminoacyl-tRNAs, that 14C-labeled aminoacyl-tRNAs synthesized endogenously from the free amino acids are preferentially sequestered within the cell, whereas their exogenous 3H counterparts distribute between the inside and outside of the cell based solely on the relative volumes of the two compartments. Furthermore, the endogenous 14C-labeled aminoacyl-tRNA population is resistant to pancreatic ribonuclease action, whereas the 3H molecules are rapidly degraded. We conclude, based on these observations, that aminoacyl-tRNAs synthesized in vivo are continually associated with components of the protein synthesis machinery and are thereby retained within the permeabilized cell and are also protected from RNase action. These data provide independent evidence for the channeling model of protein biosynthesis.

Channeling of aminoacyl-tRNA for protein synthesis in vivo.

Channeling, the direct transfer of metabolic intermediates from one enzyme to another in a pathway, has received increased attention as an explanation for the high efficiency of cellular processes. The known structural organization of the protein biosynthetic machinery, and a recent suggestion that aminoacyl-tRNAs may be channeled, has led us to devise a direct test of this possibility. By employing the technique of electroporation, conditions were established for the introduction of aminoacyl-tRNAs into Chinese hamster ovary (CHO) cells. We show, by coelectroporation of various combinations of free [14C]amino acids and [3H]aminoacyl-tRNAs, that whereas the free amino acids serve as effective precursors for protein synthesis, the exogenous aminoacyl-tRNAs are utilized poorly, if at all. The lack of incorporation into protein from added aminoacyl-tRNAs is not due to their leakage from the cell, to their instability, or to their damage during electroporation. Furthermore, in contrast to the findings with intact cells, extracts of CHO cells incorporate both free amino acids and aminoacyl-tRNAs into protein with similar efficiencies. Based on these observations, we conclude that the inability of exogenous aminoacyl-tRNAs to serve as precursors for protein synthesis is due to the structural organization of intact cells that leads to channeling of this substrate in vivo. Thus, we propose that endogenously synthesized aminoacyl-tRNA is directly transferred from aminoacyl-tRNA synthetase to elongation factor to ribosome without dissociation into the cell fluid, and as a consequence, usage of exogenously introduced molecules is precluded.

Supramolecular organization of the mammalian translation system.

Although evidence suggests that the protein synthetic machinery is organized within cells, this point has been difficult to prove because any organization that might exist is lost upon preparation of the cell-free systems usually used to study translation in vitro. To examine this process under conditions more representative of the intact cell, we have developed an active protein-synthesizing system using Chinese hamster ovary (CHO) cells permeabilized with the plant glycoside saponin. This procedure renders cells permeable to trypan blue and exogenous tRNA, but there is little release of endogenous macromolecules. Protein synthesis in this system proceeds at the same rate as that in intact cells and is about 40-fold faster than that in a cell-free system prepared from the same cells. Active protein synthesis in this system requires the addition of only Mg2+, K+, and creatine phosphate, with a small further stimulation by ATP and an amino acid mixture; no exogenous macromolecules are necessary. The proteins synthesized in this system are indistinguishable from those made by the intact cell, and the channeling of aminoacyl-tRNA observed in vivo is maintained. Our data suggest that the permeabilized cell system retains the protein-synthesizing capabilities of the intact cell and presumably its internal structure as well. Studies with this system demonstrate that the protein-synthesizing apparatus is highly organized and that its macromolecular components are not freely diffusible in mammalian cells.

Functional interaction of mammalian valyl-tRNA synthetase with elongation factor EF-1alpha in the complex with EF-1H.

In mammalian cells valyl-tRNA synthetase (ValRS) forms a high Mr complex with the four subunits of elongation factor EF-1H. The beta, gamma, and delta subunits, that contribute the guanine nucleotide exchange activity of EF-1H, are tightly associated with the NH2-terminal polypeptide extension of valyl-tRNA synthetase. In this study, we have examined the possibility that the functioning of the companion enzyme EF-1alpha could regulate valyl-tRNA synthetase activity. We show here that the addition of EF-1alpha and GTP in excess in the aminoacylation mixture is accompanied by a 2-fold stimulation of valyl-tRNAVal synthesis catalyzed by the valyl-tRNA synthetase component of the ValRS.EF-1H complex. This effect is not observed in the presence of EF-1alpha and GDP or EF-Tu.GTP and requires association of valyl-tRNA synthetase within the ValRS.EF-1H complex. Since valyl-tRNA synthetase and elongation factor EF-1alpha catalyze two consecutive steps of the in vivo tRNA cycle, aminoacylation and formation of the ternary complex EF-1alpha.GTP. Val-tRNAVal that serves as a vector of tRNA from the synthetase to the ribosome, the data suggest a coordinate regulation of these two successive reactions. The EF-1alpha.GTP-dependent stimulation of valyl-tRNA synthetase activity provides further evidence for tRNA channeling during protein synthesis in mammalian cells.

Extended conformation of mammalian translation elongation factor 1A in solution.

The conformation of mammalian elongation factor eEF1A in solution was examined by the small angle neutron scattering and scanning microcalorimetry. We have found that in contrast to the bacterial analogue the eEF1A molecule has no fixed rigid structure in solution. The radius of gyration of the eEF1A molecule (5.2 nm) is much greater than that of prokaryotic EF1A. The specific heat of denaturation is considerably lower for eEF1A than for EF1A, suggesting that the eEF1A conformation is significantly more disordered. Despite its flexible conformation, eEF1A is found to be highly active in different functional tests. According to the neutron scattering data, eEF1A becomes much more compact in the complex with uncharged tRNA. The absence of a rigid structure and the possibility of large conformational change upon interaction with a partner molecule could be important for eEF1A functioning in channeled protein synthesis and/or for the well-known capability of the protein to interact with different ligands besides the translational components.