Tagging of functional ribosomes in living cells by HaloTag® technology.

Ribosomal proteins and ribosomal associated proteins are complicated subjects to target and study because of their high conservation through evolution which led to highly structured and regulated proteins. Tagging of ribosomal proteins may allow following of protein synthesis in vivo and isolating translated mRNAs. HaloTag® is a new technology which allows detection in living cells, biochemical purification, and localization studies. In the present work, we tested HaloTag®-based ribosomal tagging. We focused on eIF6 (eukaryotic Initiation Factor 6 free 60S ribosomal marker), RACK1 (Receptor for Activated C Kinase 1; 40S and polysomes, not nuclear), and rpS9 (40S ribosomes, both in the nucleus and in the cytoplasm). Experiments performed on HEK293 cells included ribosomal profiles and Western blot on the fractions, purification of HaloTag® proteins, and fluorescence with time-lapse microscopy. We show that tagged proteins can be incorporated on ribosomes and followed by time-lapse microscopy. eIF6 properly accumulates in the nucleolus, and it is redistributed upon actinomycin D treatment. RACK1 shows a specific cytoplasmic localization, whereas rpS9 is both nucleolar and cytoplasmic. However, efficiency of purification varies due to steric hindrances. In addition, the level of overexpression and degradation may vary upon different constructs. In summary, HaloTag® technology is highly suitable to ribosome tagging, but requires prior characterization for each construct.

TRBP homolog interacts with eukaryotic initiation factor 6 (eIF6) in Fenneropenaeus chinensis.

The HIV transactivating response RNA-binding protein (TRBP) plays an important role in many biological processes. We have cloned three cDNAs from newly identified genes in the TRBP family from Fenneropenaeus chinensis. These genes have been designated Fc-TRBP1-3. Recombinant Fc-TRBP1, which was produced in Escherichia coli, was used for panning of a T7 phage display library of the Chinese shrimp hemocytes. From this panning, Fc-eukaryotic initiation factor 6 (Fc-eIF6) was isolated and sequenced. Fc-eIF6 was then cloned, recombinantly expressed, and shown to interact with Fc-TRBP by the performance of pull-down assays and Far Western blot analysis. Expression of Fc-TRBP was detected in many tissues, with elevated expression in the heart, gill, and intestine in the early stages of infection by the white spot syndrome virus (WSSV), and enhanced expression in most tissues following challenge with Vibrio anguillarum. Western blot studies confirmed the increased expression of Fc-TRBP in the gill after WSSV infection. The expression pattern of eIF6 was also analyzed and its expression was also up-regulated in intestine of WSSV-challenged shrimp. The replication of WSSV was reduced after injection of Fc-TRBP. These results indicate that Fc-TRBP and Fc-eIF6 may be components of the RNA-induced silencing complex (RISC), and thereby play a crucial role in the antiviral defense response of shrimp.

In vitro studies of archaeal translational initiation.

Initiation is the step of translation that has incurred the greatest evolutionary divergence. In silico and experimental studies have shown that archaeal translation initiation resembles neither the bacterial nor the eukaryotic paradigm, but shares features with both. The structure of mRNA in archaea is similar to the bacterial one, although the protein factors that assist translational initiation are more numerous than in bacteria and are homologous to eukaryotic proteins. This chapter describes a number of techniques that can be used for in vitro studies of archaeal translation and translational initiation, using as a model system the thermophilic crenarcheon Sulfolobus solfataricus, growing optimally at about 80 degrees in an acidic environment.

Functional analysis of the translation factor aIF2/5B in the thermophilic archaeon Sulfolobus solfataricus.

The protein IF2/eIF5B is one of the few translation initiation factors shared by all three primary domains of life (bacteria, archaea, eukarya). Despite its phylogenetic conservation, the factor is known to present marked functional divergences in the bacteria and the eukarya. In this work, the function in translation of the archaeal homologue (aIF2/5B) has been analysed in detail for the first time using a variety of in vitro assays. The results revealed that the protein is a ribosome-dependent GTPase which strongly stimulates the binding of initiator tRNA to the ribosomes even in the absence of other factors. In agreement with this finding, aIF2/5B enhances the translation of both leadered and leaderless mRNAs when expressed in a cell-free protein-synthesizing system. Moreover, the degree of functional conservation of the IF2-like factors in the archaeal and bacterial lineages was investigated by analysing the behaviour of 'chimeric' proteins produced by swapping domains between the Sulfolobus solfataricus aIF2/5B factor and the IF2 protein of the thermophilic bacterium Bacillus stearothermophilus. Beside evidencing similarities and differences between the archaeal and bacterial factors, these experiments have provided insight into the common role played by the IF2/5B proteins in all extant cells.

Modification of eukaryotic initiation factor 5A from Plasmodium vivax by a truncated deoxyhypusine synthase from Plasmodium falciparum: An enzyme with dual enzymatic properties.

The increasing resistance of the malaria parasites enforces alternative directions in finding new drug targets. Present findings from the malaria parasite Plasmodium vivax, causing tertiary malaria, suggest eukaryotic initiation factor 5A (eIF-5A) to be a promising target for the treatment of malaria. Previously we presented the 162 amino acid sequence of eukaryotic initiation factor 5A (eIF-5A) from Plasmodium vivax. In the present study, we have expressed and purified the 20kDa protein performed by one-step Nickel chelate chromatography. In Western blot experiments eIF-5A from P. vivax crossreacts with a polyclonal anti-eIF-5A antiserum from the plant Nicotiana plumbaginifolia (Solanaceae). Transcription of eIF-5A can be observed in both different developmental stages of the parasite being prominent in trophozoites. We recently published the nucleic acid sequence from a genomic clone of P. falciparum strain NF54 encoding a putative deoxyhypusine synthase (DHS), an enzyme that catalyzes the post-translational modification of eIF-5A. After removal of 22 amino acids DHS was expressed as a Histidin fusion protein and purified by Nickel affinity chromatography. Truncated DHS from P. falciparum modifies eIF-5A from P. vivax. DHS from P. falciparum NF54 is a bi-functional protein with dual enzymatic specificities, that is, DHS activity and homospermidine synthase activity (HSS) (0.047 pkatal/mg protein) like in other eukaryotes. Inhibition of DHS from P. falciparum resulted in a K(i) of 0.1 microM for the inhibitor GC7 being 2000-fold less than the nonguanylated derivative 1,7-diaminoheptane. Dhs transcription occurs in both develomental stages suggesting its necessity in cell proliferation.

Purified Argonaute2 and an siRNA form recombinant human RISC.

Genetic, biochemical and structural studies have implicated Argonaute proteins as the catalytic core of the RNAi effector complex, RISC. Here we show that recombinant, human Argonaute2 can combine with a small interfering RNA (siRNA) to form minimal RISC that accurately cleaves substrate RNAs. Recombinant RISC shows many of the properties of RISC purified from human or Drosophila melanogaster cells but also has surprising features. It shows no stimulation by ATP, suggesting that factors promoting product release are missing from the recombinant enzyme. The active site is made up of a unique Asp-Asp-His (DDH) motif. In the RISC reconstitution system, the siRNA 5' phosphate is important for the stability and the fidelity of the complex but is not essential for the creation of an active enzyme. These studies demonstrate that Argonaute proteins catalyze mRNA cleavage within RISC and provide a source of recombinant enzyme for detailed biochemical studies of the RNAi effector complex.

The archaeal eIF2 homologue: functional properties of an ancient translation initiation factor.

The eukaryotic translation initiation factor 2 (eIF2) is pivotal for delivery of the initiator tRNA (tRNAi) to the ribosome. Here, we report the functional characterization of the archaeal homologue, a/eIF2. We have cloned the genes encoding the three subunits of a/eIF2 from the thermophilic archaeon Sulfolobus solfataricus, and have assayed the activities of the purified recombinant proteins in vitro. We demonstrate that the trimeric factor reconstituted from the recombinant polypeptides has properties similar to those of its eukaryal homologue: it interacts with GTP and Met-tRNAi, and stimulates binding of the latter to the small ribosomal subunit. However, the archaeal protein differs in some functional aspects from its eukaryal counterpart. In contrast to eIF2, a/eIF2 has similar affinities for GDP and GTP, and the beta-subunit does not contribute to tRNAi binding. The detailed analysis of the complete trimer and of its isolated subunits is discussed in light of the evolutionary history of the eIF2-like proteins.

Identification of mRNA that binds to eukaryotic initiation factor 5A by affinity co-purification and differential display.

Eukaryotic initiation factor 5A (eIF-5A) is the only protein in nature that contains hypusine, an unusual amino acid formed post-translationally by deoxyhypusine synthase and deoxyhypusine hydroxylase. Genetic and pharmacological evidence suggests that eIF-5A is essential for cell survival and proliferation. However, the precise function and interacting partners of eIF-5A remain unclear. We have shown previously that eIF-5A can bind to RRE (Rev-response element) and U6 RNA in vitro. Using SELEX (systematic evolution of ligands by exponential enrichment), we have also shown that eIF-5A is capable of binding to RNA in a sequence-specific manner [Xu and Chen (2001) J. Biol. Chem. 276, 2555-2561]. In the present paper, we show that the identification of mRNA species that bind to eIF-5A can be achieved by affinity co-purification and PCR differential display. Using this approach with three sets of anchoring and arbitrary primers, we have found 20 RNA sequences that co-purified specifically with eIF-5A. Five of them contained AAAUGU, the putative eIF-5A-interacting element that we identified previously using the SELEX method. Direct binding of the cloned RNA to eIF-5A could be demonstrated by electrophoretic mobility-shift assay. BLAST analysis revealed that the eIF-5A-interacting RNAs encode proteins such as ribosomal L35a, plasminogen activation inhibitor mRNA-binding protein, NADH dehydrogenase subunit and ADP-ribose pyrophosphatase. Some, however, encode hypothetical proteins. All the cloned RNAs have the potential to form extensive stem-loop structures.

Screening assay for the identification of deoxyhypusine synthase inhibitors.

The 1st step in the posttranslational hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)lysine] modification of eukaryotic translation initiation factor 5A (eIF5A) is catalyzed by deoxyhypusine synthase (DHS). The eIF5A intermediate is subsequently hydroxylated by deoxyhypusine hydroxylase (DHH), thereby converting the eIF5A precursor into a biologically active protein. Depletion of eIF5A causes inhibition of cell growth, and the identification of eIF5A as a cofactor of the HIV Rev protein turns this host protein and therefore DHS into an interesting target for drugs against abnormal cell growth and/or HIV replication. The authors developed a 96-well format DHS assay applicable for the screening of DHS inhibitors. Using this assay, they demonstrate DHS inhibition by AXD455 (Semapimod, CNI-1493). This assay represents a powerful tool for the identification of new DHS inhibitors with potency against cancer and HIV.

Translation initiation factor eIF-5A from Plasmodium falciparum.

Eukaryotic translation initiation factor (eIF-5A) is a highly conserved and essential protein that contains the unique amino acid hypusine. The first step in the post-translational biosynthesis of hypusine, the transfer of an aminobutyl moiety from the polyamine substrate spermidine to the -amino group of a specific lysine residue in the eIF-5A precursor, is catalyzed by the enzyme deoxyhypusine synthase. A cDNA encoding a protein homologous to eIF-5A was isolated by plaque hybridization from a cDNA library of Plasmodium falciparum. The cloned cDNA contains an open reading frame encoding a protein of 161 amino acids, which shares a high sequence identity with other eukaryotic eIF-5A sequences. A phylogenetic tree constructed with eIF-5A from P. falciparum and 16 other eIF-5A sequences of eukaryotic and archaeal origin reveals that plasmodial eIF-5A together with other apicomplexan eIF-5A show a higher degree of homology to plant proteins than to animal and fungal sequences. The plasmodial eIF-5A gene was expressed as a six-histidine tagged fusion protein in Escherichia coli. Radioactive incorporation studies with [1,8-3H] spermidine indicated that this protein can serve as a substrate for human deoxyhypusine synthase. Results of quantitative real-time PCR studies with synchronized erythrocytic stages of P. falciparum revealed no significant induction or downregulation but only some variation in the expression level of plasmodial eIF-5A in ring, trophozoite and schizont stage.

Higher activity of recombinant bovine deoxyhypusine synthase vs. human deoxyhypusine synthase.

Mature eukaryotic initiation factor 5A (eIF5A) is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine (Nepsilon-(4-amino-2(R)-hydroxybutyl)lysine). The synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Deoxyhypusine synthase is the first of the two enzymes that catalyzes the maturation of eIF5A. We have subcloned the cDNA encoding bovine and human deoxyhypusine synthase into a pET-11a expression vector, separately. T7-tagged bovine and human deoxyhypusine synthase have been overexpressed in Escherichia coli and purified to homogeneity using T7 antibody affinity chromatography. Activities of the enzyme from both human and bovine have been measured by their ability to convert the eIF5A precursor protein to the intermediate, deoxyhypusine form of eIF5A. Our results have shown that bovine deoxyhypusine synthase has considerably higher activity than human deoxyhypusine synthase in catalyzing the synthesis of deoxyhypusine.

Phosphorylation of mammalian eukaryotic translation initiation factor 6 and its Saccharomyces cerevisiae homologue Tif6p: evidence that phosphorylation of Tif6p regulates its nucleocytoplasmic distribution and is required for yeast cell growth.

The synthesis of 60S ribosomal subunits in Saccharomyces cerevisiae requires Tif6p, the yeast homologue of mammalian eukaryotic translation initiation factor 6 (eIF6). In the present work, we have isolated a protein kinase from rabbit reticulocyte lysates on the basis of its ability to phosphorylate recombinant human eIF6. Mass spectrometric analysis as well as antigenic properties of the purified kinase identified it as casein kinase I. The site of in vitro phosphorylation, which is highly conserved from yeast to mammals, was identified as the serine residues at positions 174 (major site) and 175 (minor site). The homologous yeast protein Tif6p was also phosphorylated in vivo in yeast cells. Mutation of Tif6p at serine-174 to alanine reduced phosphorylation drastically and caused loss of cell growth and viability. When both Ser-174 and Ser-175 were mutated to alanine, phosphorylation of Tif6p was completely abolished. Furthermore, while wild-type Tif6p was distributed both in nuclei and the cytoplasm of yeast cells, the mutant Tif6p (with Ser174Ala and Ser175Ala) became a constitutively nuclear protein. These results suggest that phosphorylatable Ser-174 and Ser-175 play a critical role in the nuclear export of Tif6p.

RNA-binding activity of translation initiation factor eIF4G1 from Saccharomyces cerevisiae.

We identified and mapped RNA-binding sites of yeast Saccharomyces cerevisiae translation initiation factor eIF4G1 and examined their importance for eIF4G1 function in vitro and in vivo. Yeast eIF4G1 binds to single-stranded RNA with three different sites, the regions of amino acids 1-82 (N terminus), 492-539 (middle), and 883-952 (C terminus). The middle and C-terminal RNA-binding sites represent RS (arginine and serine)-rich domains; the N-terminal site is asparagine-, glutamine- and glycine-rich. The three RNA-binding sites have similar affinity for single-stranded RNA, whereas the affinity for single-stranded RNA full-length eIF4G1 is about 100-fold higher (approximate K(d) of 5 x 10(-8) M). Replacement of the arginine residues in the middle RS site by alanine residues abolishes its RNA-binding activity. Deletion of individual RNA-binding sites shows that eIF4G1 molecules lacking one binding site are still active in supporting growth of yeast cells and translation in vitro, whereas eIF4G1 molecules lacking two or all three RNA-binding sites are strongly impaired or inactive. These data suggest that RNA-binding activity is required for eIF4G1 function.

Mammalian translation initiation factor eIF1 functions with eIF1A and eIF3 in the formation of a stable 40 S preinitiation complex.

We have examined the role of the mammalian initiation factor eIF1 in the formation of the 40 S preinitiation complex using in vitro binding of initiator Met-tRNA (as Met-tRNA(i).eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA. We observed that, although both eIF1A and eIF3 are essential to generate a stable 40 S preinitiation complex, quantitative binding of the ternary complex to 40 S subunits also required eIF1. The 40 S preinitiation complex contained, in addition to eIF3, both eIF1 and eIF1A in a 1:1 stoichiometry with respect to the bound Met-tRNA(i). These three initiation factors also bind to free 40 S subunits, and the resulting complex can act as an acceptor of the ternary complex to form the 40 S preinitiation complex (40 S.eIF3.eIF1.eIF1A.Met-tRNA(i).eIF2.GTP). The stable association of eIF1 with 40 S subunits required the presence of eIF3. In contrast, the binding of eIF1A to free 40 S ribosomes as well as to the 40 S preinitiation complex was stabilized by the presence of both eIF1 and eIF3. These studies suggest that it is possible for eIF1 and eIF1A to bind the 40 S preinitiation complex prior to mRNA binding.

Identification of metastasis-associated genes in early stage non-small cell lung cancer by subtractive hybridization.

Non-small cell lung cancer (NSCLC) is a leading cause of death and a substantial fraction of patients with surgically resected disease ultimately dies due to distant metastasis. To identify gene expression differences in early stage adenocarcinoma that either did or did not metastasize within a 5-year period, we employed a subtractive hybridization strategy of pooled RNA from primary adenocarcinomas (stage I) of the lung. Individual clones (n=225) of the subtracted cDNA library were sequenced. Further analyses of mRNA expression levels in a cohort of 70 NSCLC patients (stage I to IIIA) showed that the metastasis association of the identified genes was stage and histology specific. Cox regression analyses identified two genes (EIF4A1, MALA1) to be independent prognostic parameters for patients' survival in stage I and II disease. These findings could help to identify early-stage NSCLC patients at high risk for the development of distant metastasis.

Development and characterization of a reconstituted yeast translation initiation system.

To provide a bridge between in vivo and in vitro studies of eukaryotic translation initiation, we have developed a reconstituted translation initiation system using components from the yeast Saccharomyces cerevisiae. We have purified a minimal set of initiation factors (elFs) that, together with yeast 80S ribosomes, GTP, and initiator methionyl-tRNA, are sufficient to assemble active initiation complexes on a minimal mRNA template. The kinetics of various steps in the pathway of initiation complex assembly and the formation of the first peptide bond in vitro have been explored. The formation of active initiation complexes in this system is dependent on ribosomes, mRNA, Met-tRNAi, GTP hydrolysis, elF1, elF1A, elF2, elF5, and elF5B. Our data indicate that elF1 and elF1A both facilitate the binding of the elF2 x GTP x Met-tRNAi complex to the 40S ribosomal subunit to form the 43S complex. elF5 stimulates a step after 43S complex formation, consistent with its proposed role in activating GTP hydrolysis by elF2 upon initiation codon recognition. The presence of elF5B is required for the joining of the 40S and 60S subunits to form the 80S initiation complex. The step at which each of these factors acts in this reconstituted system is in agreement with previous data from in vivo studies and work using reconstituted mammalian systems, indicating that the system recapitulates fundamental events in translation initiation in eukaryotic cells. This system should allow us to couple powerful yeast genetic and molecular biological experiments with in vitro kinetic and biophysical experiments, yielding a better understanding of the molecular mechanics of this central, complex process.

A subcomplex of three eIF3 subunits binds eIF1 and eIF5 and stimulates ribosome binding of mRNA and tRNA(i)Met.

Yeast translation initiation factor 3 contains five core subunits (known as TIF32, PRT1, NIP1, TIF34 and TIF35) and a less tightly associated component known as HCR1. We found that a stable subcomplex of His8-PRT1, NIP1 and TIF32 (PN2 subcomplex) could be affinity purified from a strain overexpressing these eIF3 subunits. eIF5, eIF1 and HCR1 co-purified with this subcomplex, but not with distinct His8-PRT1- TIF34-TIF35 (P45) or His8-PRT1-TIF32 (P2) sub complexes. His8-PRT1 and NIP1 did not form a stable binary subcomplex. These results provide in vivo evidence that TIF32 bridges PRT1 and NIP1, and that eIFs 1 and 5 bind to NIP1, in native eIF3. Heat-treated prt1-1 extracts are defective for Met-tRNA(i)Met binding to 40S subunits, and we also observed defective 40S binding of mRNA, eIFs 1 and 5 and eIF3 itself in these extracts. We could rescue 40S binding of Met- tRNA(i)Met and mRNA, and translation of luciferase mRNA, in a prt1-1 extract almost as well with purified PN2 subcomplex as with five-subunit eIF3, whereas the P45 subcomplex was nearly inactive. Thus, several key functions of eIF3 can be carried out by the PRT1-TIF32-NIP1 subcomplex.

Further characterization of the helicase activity of eIF4A. Substrate specificity.

Eukaryotic initiation factor (eIF) 4A is the archetypal member of the DEAD box family of RNA helicases and is proposed to unwind structures in the 5'-untranslated region of mRNA to facilitate binding of the 40 S ribosomal subunit. The helicase activity of eIF4A has been further characterized with respect to substrate specificity and directionality. Results confirm that the initial rate and amplitude of duplex unwinding by eIF4A is dependent on the overall stability, rather than the length or sequence, of the duplex substrate. eIF4A helicase activity is minimally dependent on the length of the single-stranded region adjacent to the double-stranded region of the substrate. Interestingly, eIF4A is able to unwind blunt-ended duplexes. eIF4A helicase activity is also affected by substitution of 2'-OH (RNA) groups with 2'-H (DNA) or 2'-methoxyethyl groups. These observations, taken together with results from competitive inhibition experiments, suggest that eIF4A may interact directly with double-stranded RNA, and recognition of helicase substrates occurs via chemical and/or structural features of the duplex. These results allow for refinement of a previously proposed model for the mechanism of action of eIF4A helicase activity.

The promyelocytic leukemia (PML) protein suppresses cyclin D1 protein production by altering the nuclear cytoplasmic distribution of cyclin D1 mRNA.

The majority of the promyelocytic leukemia (PML) protein is present in nuclear bodies which are altered in several pathogenic conditions including acute promyelocytic leukemia. PML nuclear bodies are found in nearly all cells yet their function remains unknown. Here, we demonstrate that PML and the eukaryotic initiation factor 4E (elF-4E) co-localize and co-immunopurify. eIF-4E is involved in nucleocytoplasmic transport of specific mRNAs including cyclin D1. eIF-4E overexpression leads to increased cyclin D1 protein levels; whereas, overexpression of PML leads to decreased cyclin D1 levels. Neither PML nor eIF-4E cause significant changes in cyclin D1 mRNA levels. The association with eIF-4E led us to investigate if PML could alter mRNA distribution as a possible post-transcriptional mechanism for suppressing cyclin D1 production. We show that overexpression of PML results in nuclear retention of cyclin D1 mRNA and that intact PML nuclear bodies are required. Addition of eIF-4E overcomes PML induced retention and alters the morphology of PML bodies suggesting a mechanism by which eIF-4E can modulate PML function. These results raise the possibility that PML nuclear bodies may participate in the regulation of nucleocytoplasmic transport of specific mRNAs.