Cloning of Dictyostelium eIF6 (p27) and mapping its nucle(ol)ar localization subdomains

Eukaryotic translation initiation factor 6 (eIF6), also termed p27^BBP, is an evolutionary conserved regulator of ribosomal function. The protein is involved in maturation and/or export from the nucleus of the 60S ribosomal subunit. Regulated binding to and release from the 60S subunit also regulates formation of 80S ribosomes, and thus translation. The protein is also found in hemidesmosomes of epithelial cells expressing β4 integrin and is assumed to regulate cross-talk between β4 integrin, intermediate filaments and ribosomes. In the present study we show that the Dictyostelium eIF6 (also called p27^BBP) gene is expressed during growth, down-regulated during the first hours of starvation, and up-regulated again at the end of aggregation. Phagocytosis, and to a lesser extent pinocytic uptake of axenic medium, stimulate gene expression in starving cells. The eIF6 gene is present in single copy and its ablation is lethal. We utilized the green fluorescent protein (GFT) as fusion protein marker to investigate sequences responsible for eIF6 subcellular localization. The protein is found both in cytoplasm and nucleus, and is enriched in nucleoli. Deletion sequence analysis shows that nucle(ol)ar localization sequences are located within the N- and C-terminal subdomains of the protein.     

The beta4 integrin interactor p27(BBP/eIF6) is an essential nuclear matrix protein involved in 60S ribosomal subunit assembly

p27(BBP/eIF6) is an evolutionarily conserved protein that was originally identified as p27(BBP), an interactor of the cytoplasmic domain of integrin beta4 and, independently, as the putative translation initiation factor eIF6. To establish the in vivo function of p27(BBP/eIF6), its topographical distribution was investigated in mammalian cells and the effects of disrupting the corresponding gene was studied in the budding yeast, Saccharomyces cerevisiae. In epithelial cells containing beta4 integrin, p27(BBP/eIF6) is present in the cytoplasm and enriched at hemidesmosomes with a pattern similar to that of beta4 integrin. Surprisingly, in the absence and in the presence of the beta4 integrin subunit, p27(BBP/eIF6) is in the nucleolus and associated with the nuclear matrix. Deletion of the IIH S. cerevisiae gene, encoding the yeast p27(BBP/eIF6) homologue, is lethal, and depletion of the corresponding gene product is associated with a dramatic decrease of the level of free ribosomal 60S subunit. Furthermore, human p27(BBP/eIF6) can rescue the lethal effect of the iihDelta yeast mutation. The data obtained in vivo suggest an evolutionarily conserved function of p27(BBP/eIF6) in ribosome biogenesis or assembly rather than in translation. A further function related to the beta4 integrin subunit may have evolved specifically in higher eukaryotic cells.     

Expression of p27BBP/eIF6 is highly modulated during Xenopus laevis embryogenesis

Protein p27BBP/eIF6 is necessary for ribosomal function of all cells. Previous data showed that from mammals to yeast p27BBP/eIF6 is involved in the biogenesis of ribosomal subunit 60S and its association with the 60S prevents premature 80S formation regulated by PKC signaling, indicating that phosphorylation of p27BBP/eIF6 is needed for translation to occur. While in vitro p27BBP/eIF6 is constitutively expressed, and it has a high level of expression in cycling cells, in vivo its expression varies according to tissues and appears regulated by factors up to now unknown. p27BBP/eIF6 has never been investigated in developing organisms where its upregulation can be correlated with tissue growth and differentiation. In this study we have sequenced p27BBP/eIF6 cDNA and studied its expression during development of Xenopus laevis, as the first step for studying its regulation. The amino acid sequence is highly conserved with two putative PKC phosphorylation sites in serine, one site being typical of Xenopus. At the end of gastrulation, the p27BBP/eIF6 riboprobe localizes in the neural plate and in the paraxial mesoderm. In particular, from stage 24, a clear-cut localization occurs in the perspective head. In embryos exposed to teratogens, the localization of p27BBP/eIF6 riboprobe varies according to the change of head size caused by the treatment. p27BBP/eIF6 expression is particularly evident in differentiating olfactory pits, the lens, otic vesicles, and in branchial arches. Features of particular interest are p27BBP/eIF6 high level of expression in the eye field, and in the mid-hindbrain-boundary, two regions with high proliferative activity. Altogether, data indicate that a modulated expression of p27BBP/eIF6 occurs in developing anlagens in addition to a basal level of expression, and may suggest a correlation between p27BBP/eIF6 and proliferative activity. Moreover, the X. laevis cDNA isolation and characterization offer new hints for further studies in relation to potential p27BBP/eIF6 phosphorylation.     

Evidence for alternate splicing within the mRNA transcript encoding the DNA damage response kinase ATR

The ITGB4BP gene encodes for a highly conserved protein, named p27BBP (also known as eIF6), originally identified in mammals as a cytoplasmic interactor of beta4 integrin. In vitro and in vivo studies demonstrated that p27BBP is essential for cell viability and has a primary function in the biogenesis of the 60S ribosomal subunit. Here we report the genomic organization of the human ITGB4BP gene and show that its gene product is expressed with features of a housekeeping element in vitro, but is regulated in a cell specific fashion in vivo. The human gene spans 10 kb and comprises seven exons and six introns. The 5' flanking region shows a TATA-less promoter, canonical CpG islands, and binding sites for serum responsive elements. In cultured cells, p27BBP mRNA and protein are constitutively expressed and stable. A gradual loss of p27BBP mRNA can be observed only after prolonged serum starvation, and heat shock treatment. In contrast, p27BBP mRNA and protein levels in vivo are variable among different organs. More strikingly, immunohistochemical analysis shows that the p27BBP protein is present in a cell specific fashion, even within the same tissue. Taken together, these data show that ITGB4BP gene expression is highly regulated in vivo, possibly by the combination of tissue specific factors and protein synthesis pathways.     

The Saccharomyces cerevisiae Homologue of Mammalian Translation Initiation Factor 6 Does Not Function as a Translation Initiation Factor

Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. The Saccharomyces cerevisiae gene that encodes the 245-amino-acid eIF6 (calculated M_r 25,550), designated TIF6, has been cloned and expressed in Escherichia coli. The purified recombinant protein prevents association between 40S and 60S ribosomal subunits to form 80S ribosomes. TIF6 is a single-copy gene that maps on chromosome XVI and is essential for cell growth. eIF6 expressed in yeast cells associates with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Depletion of eIF6 from yeast cells resulted in a decrease in the rate of protein synthesis, accumulation of half-mer polyribosomes, reduced levels of 60S ribosomal subunits resulting in the stoichiometric imbalance in the 40S/60S subunit ratio, and ultimately cessation of cell growth. Furthermore, lysates of yeast cells depleted of eIF6 remained active in translation of mRNAs in vitro. These results indicate that eIF6 does not act as a true translation initiation factor. Rather, the protein may be involved in the biogenesis and/or stability of 60S ribosomal subunits.     

Formation of nuclear matrix filaments by p27(BBP)/eIF6

p27(BBP)/eIF6 is an evolutionarily conserved protein necessary for ribosome biogenesis which was cloned in mammals for its ability to bind the cytodomain of beta 4 integrin. In cultured cells, a conspicuous fraction of p27(BBP)/eIF6 is associated with the intermediate filaments/nuclear matrix (IF/NM) cytoskeleton. The mechanism of this association is not known. Here we show that in epidermis p27(BBP)/eIF6 is naturally associated with IF/NM. To analyze the intrinsic capability of p27(BBP)/eIF6 to generate cytoskeletal networks, the properties of the pure, recombinant, untagged protein were studied. Recombinant p27(BBP)/eIF6 binds beta 4 integrin. Upon dialysis against IF buffer, p27(BBP)/eIF6 forms polymers which, strikingly, have a morphology identical to NM filaments. Cross-linking experiments suggested that polymerization is favored by the formation of disulphide bridges. These data suggest that p27(BBP)/eIF6 is associated with the cytoskeleton, and contributes to formation of NM filaments. These findings help to settle the controversy on nuclear matrix.     

Function and ribosomal localization of aIF6, a translational regulator shared by archaea and eukarya

The translation factor IF6 is shared by the Archaea and the Eukarya, but is not found in Bacteria. The properties of eukaryal IF6 (eIF6) have been extensively studied, but remain somewhat elusive. eIF6 behaves as a ribosome-anti-association factor and is involved in miRNA-mediated gene silencing; however, it also seems to participate in ribosome synthesis and export. Here we have determined the function and ribosomal localization of the archaeal (Sulfolobus solfataricus) IF6 homologue (aIF6). We find that aIF6 binds specifically to the 50S ribosomal subunits, hindering the formation of 70S ribosomes and strongly inhibiting translation. aIF6 is uniformly expressed along the cell cycle, but it is upregulated following both cold- and heat shock. The aIF6 ribosomal binding site lies in the middle of the 30-S interacting surface of the 50S subunit, including a number of critical RNA and protein determinants involved in subunit association. The data suggest that the IF6 protein evolved in the archaeal–eukaryal lineage to modulate translational efficiency under unfavourable environmental conditions, perhaps acquiring additional functions during eukaryotic evolution.     

Moe1 and spInt6, the fission yeast homologues of mammalian translation initiation factor 3 subunits p66 (eIF3d) and p48 (eIF3e), respectively, are required for stable association of eIF3 subunits

The protein encoded by the fission yeast gene, moe1(+) is the homologue of the p66/eIF3d subunit of mammalian translation initiation factor eIF3. In this study, we show that in fission yeast, Moe1 physically associates with eIF3 core subunits as well as with 40 S ribosomal particles as a constituent of the eIF3 protein complex that is similar in size to multisubunit mammalian eIF3. However, strains lacking moe1(+) (Deltamoe1) are viable and show no gross defects in translation initiation, although the rate of translation in the Deltamoe1 cells is about 30-40% slower than wild-type cells. Mutant Deltamoe1 cells are hypersensitive to caffeine and defective in spore formation. These phenotypes of Deltamoe1 cells are similar to those reported previously for deletion of the fission yeast int6(+) gene that encodes the fission yeast homologue of the p48/Int6/eIF3e subunit of mammalian eIF3. Further analysis of eIF3 subunits in Deltamoe1 or Deltaint6 cells shows that in these deletion strains, while all the eIF3 subunits are bound to 40 S particles, dissociation of ribosome-bound eIF3 results in the loss of stable association between the eIF3 subunits. In contrast, eIF3 isolated from ribosomes of wild-type cells are associated with one another in a protein complex. These observations suggest that Moe1 and spInt6 are each required for stable association of eIF3 subunits in fission yeast.     

The Saccharomyces cerevisiae TIF6 gene encoding translation initiation factor 6 is required for 60S ribosomal subunit biogenesis

Eukaryotic translation initiation factor 6 (eIF6), a monomeric protein of about 26 kDa, can bind to the 60S ribosomal subunit and prevent its association with the 40S ribosomal subunit. In Saccharomyces cerevisiae, eIF6 is encoded by a single-copy essential gene. To understand the function of eIF6 in yeast cells, we constructed a conditional mutant haploid yeast strain in which a functional but a rapidly degradable form of eIF6 fusion protein was synthesized from a repressible GAL10 promoter. Depletion of eIF6 from yeast cells resulted in a selective reduction in the level of 60S ribosomal subunits, causing a stoichiometric imbalance in 60S-to-40S subunit ratio and inhibition of the rate of in vivo protein synthesis. Further analysis indicated that eIF6 is not required for the stability of 60S ribosomal subunits. Rather, eIF6-depleted cells showed defective pre-rRNA processing, resulting in accumulation of 35S pre-rRNA precursor, formation of a 23S aberrant pre-rRNA, decreased 20S pre-rRNA levels, and accumulation of 27SB pre-rRNA. The defect in the processing of 27S pre-rRNA resulted in the reduced formation of mature 25S and 5.8S rRNAs relative to 18S rRNA, which may account for the selective deficit of 60S ribosomal subunits in these cells. Cell fractionation as well as indirect immunofluorescence studies showed that c-Myc or hemagglutinin epitope-tagged eIF6 was distributed throughout the cytoplasm and the nuclei of yeast cells.     

Zinc finger-like motifs in rat ribosomal proteins S27 and S29.

The primary structures of the rat 40S ribosomal subunit proteins S27 and S29 were deduced from the sequences of nucleotides in recombinant cDNAs and confirmed by determination of amino acid sequences in the proteins. Ribosomal protein S27 has 83 amino acids and the molecular weight is 9,339. Hybridization of cDNA to digests of nuclear DNA suggests that there are 4-6 copies of the S27 gene; the mRNA for the protein is about 620 nucleotides in length. Ribosomal protein S29 has 55 amino acids and the molecular weight is 6,541. There are 14-17 copies of the S29 gene and its mRNA is about 500 nucleotides in length. Rat ribosomal protein S29 is related to several members of the archaebacterial and eubacterial S14 family of ribosomal proteins. S27 and S29 have zinc finger-like motifs as do other proteins from eukaryotic, archaebacterial, eubacterial, and mitochondrial ribosomes. Moreover, ribosomes and ribosomal subunits appear to contain zinc and iron as well.     

Position of eukaryotic initiation factor eIF5B on the 80S ribosome mapped by directed hydroxyl radical probing

Eukaryotic translation initiation factor eIF5B is a ribosome-dependent GTPase that mediates displacement of initiation factors from the 40S ribosomal subunit in 48S initiation complexes and joining of 40S and 60S subunits. Here, we determined eIF5B's position on 80S ribosomes by directed hydroxyl radical cleavage. In the resulting model, eIF5B is located in the intersubunit cleft of the 80S ribosome: domain 1 is positioned near the GTPase activating center of the 60S subunit, domain 2 interacts with the 40S subunit (helices 3, 5 and the base of helix 15 of 18S rRNA and ribosomal protein (rp) rpS23), domain 3 is sandwiched between subunits and directly contacts several ribosomal elements including Helix 95 of 28S rRNA and helix 44 of 18S rRNA, domain 4 is near the peptidyl-transferase center and its helical subdomain contacts rpL10E. The cleavage data also indicate that binding of eIF5B might induce conformational changes in both subunits, with ribosomal segments wrapping around the factor. Some of these changes could also occur upon binding of other translational GTPases, and may contribute to factor recognition.     

Translation Initiation Factor eIF4B Interacts with a Picornavirus Internal Ribosome Entry Site in both 48S and 80S Initiation Complexes Independently of Initiator AUG Location

Most eukaryotic initiation factors (eIFs) are required for internal translation initiation at the internal ribosome entry site (IRES) of picornaviruses. eIF4B is incorporated into ribosomal 48S initiation complexes with the IRES RNA of foot-and-mouth disease virus (FMDV). In contrast to the weak interaction of eIF4B with capped cellular mRNAs and its release upon entry of the ribosomal 60S subunit, eIF4B remains tightly associated with the FMDV IRES during formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. The interaction of eIF4B with the IRES in 48S and 80S complexes is independent of the location of the initiator AUG and thus independent of the mechanism by which the small ribosomal subunit is placed at the actual start codon, either by direct internal ribosomal entry or by scanning. eIF4B does not greatly rearrange its binding to the IRES upon entry of the ribosomal subunits, and the interaction of eIF4B with the IRES is independent of the polypyrimidine tract-binding protein, which enhances FMDV translation.     

Changes in ribosomal binding activity of eIF3 correlate with increased translation rates during activation of T lymphocytes

The rate of protein synthesis in quiescent peripheral blood T lymphocytes increases dramatically following mitogenic activation. The stimulation of translation is due to an increase in the rate of initiation caused by the regulation of initiation factor activities. Here, we focus on eIF3, a large multiprotein complex that plays a central role in the formation of the 40 S initiation complex. Using sucrose density gradient centrifugation to analyze ribosome complexes, we find that most eIF3 is not bound to 40 S ribosomal subunits in unactivated T lymphocytes but becomes ribosome-bound following activation. Immunoblot analyses of sucrose gradient fractions for individual eIF3 subunits show that the small eIF3j subunit is unassociated with the eIF3 complex in quiescent T lymphocytes, but upon activation joins the other eIF3 subunits and binds 40 S ribosomal subunits. Because eIF3j has been shown to be required for eIF3 binding to 40 S ribosomes in vitro, the results suggest that mitogenic stimulation of T lymphocytes leads to an activation of eIF3j, thereby enabling eIF3 to bind to the larger ribosome-free eIF3 subunit complex, and then to the 40 S ribosomes. The association of eIF3j with the other eIF3 subunits appears to be inhibited by rapamycin, suggesting a mechanism that lies downstream from the mammalian target of rapamycin kinase. This association requires ionomycin together with a phorbol ester, which also suggests that calcium signaling is involved. We conclude that the complex formation of eIF3 and its association with the ribosomes might contribute to increased translation rates during T lymphocyte activation.     

Translational control by 80S formation and 60S availability: The central role of eIF6, a rate limiting factor in cell cycle progression and tumorigenesis

Ribosome biogenesis and translation can be simplified as the processes of generating ribosomes and their use for decoding mRNA into a protein. Ribosome biogenesis has been efficiently studied in unicellular organisms like the budding yeast, allowing us a deep and basic knowledge of this process in growing cells. Translation has been modeled in vitro and in unicellular organisms. These studies have given us an important insight into the mechanisms and evolutionarily conserved aspects of ribosome biology. However, we advocate the need of the direct study of these processes in multicellular organisms. Analysis of ribosome biogenesis and translation in vivo in Metazoa and mammalian models is emerging and unveils the unexpected consequences of perturbed ribosome biogenesis and translation. Here, we will describe how one factor, eIF6, plays a crucial role both in the generation of the large ribosomal subunit and its availability for translation. From there, we will make specific conclusions on the physiological relevance of eIF6 in 80S formation, cell cycle progression and disease, raising the point that the control of gene expression may occur at the unexpected level of the large ribosomal subunit. In the future, the modulation of eIF6 binding to the 60S may be pharmacologically exploited to reduce the growth of cancer cells or ameliorate the phenotype of SDS syndrome.     

The essential GTPase RbgA (YlqF) is required for 50S ribosome assembly in Bacillus subtilis

In this paper the essential GTPase YlqF is shown to participate in the biogenesis of the 50S ribosomal subunit in Bacillus subtilis. Cells depleted of YlqF displayed gene expression profiles and nucleoid morphologies that were consistent with a function for YlqF in translation. In addition, YlqF is evolutionarily linked to two eukaryotic GTPases, Nog2p and Nug1p, that are involved in the biogenesis and the nuclear export of the 60S ribosomal subunit. Analysis of ribosomes from cells depleted of YlqF demonstrated that the formation of 70S ribosomes was greatly reduced and the large subunit sedimented at 45S. Cells grown with varying depleted levels of YlqF, yielding doubling times ranging from 38 min to 150 min, all displayed the 45S intermediate. Purified YlqF-His(6) protein associates with the 45S intermediate, but not the mature 50S subunit in vitro. Analysis of proteins from the 45S intermediate indicated that ribosomal protein L16, which is added late during in vitro Escherichia coli 50S ribosome biogenesis, was missing from the 45S intermediate. These results support a model in which YlqF participates in the formation of active 70S ribosomes in the cell by functioning in a late step of 50S subunit biogenesis. Based on these results we propose to rename the ylqF gene rbgA (ribosome biogenesis GTPase A).     

Phosphorylation of ribosomal proteins influences subunit association and translation of poly (U) in Streptomyces coelicolor

The occurrence of phosphorylated proteins in ribosomes of Streptomyces coelicolor was investigated. Little is known about which biological functions these posttranslational modifications might fulfil. A protein kinase associated with ribosomes phosphorylated six ribosomal proteins of the small subunit (S3, S4, S12, S13, S14 and S18) and seven ribosomal proteins of the large subunit (L2, L3, L7/L12, L16, L17, L23 and L27). The ribosomal proteins were phosphorylated mainly on the Ser/Thr residues. Phosphorylation of the ribosomal proteins influences ribosomal subunits association. Ribosomes with phosphorylated proteins were used to examine poly (U) translation activity. Phosphorylation induced about 50% decrease in polyphenylalanine synthesis. After preincubation of ribosomes with alkaline phosphatase the activity of ribosomes was greatly restored. Small differences were observed between phosphorylated and unphosphorylated ribosomes in the kinetic parameters of the binding of Phe-tRNA to the A-site of poly (U) programmed ribosomes, suggesting that the initial binding of Phe-tRNA is not significantly affected by phosphorylation. On contrary, the rate of peptidyl transferase was about two-fold lower than that in unphosphorylated ribosomes. The data presented demonstrate that phosphorylation of ribosomal proteins affects critical steps of protein synthesis.     

The essential ATP-binding cassette protein RLI1 functions in translation by promoting preinitiation complex assembly

RLI1 is an essential yeast protein closely related in sequence to two soluble members of the ATP-binding cassette family of proteins that interact with ribosomes and function in translation elongation (YEF3) or translational control (GCN20). We show that affinity-tagged RLI1 co-purifies with eukaryotic translation initiation factor 3 (eIF3), eIF5, and eIF2, but not with other translation initiation factors or with translation elongation or termination factors. RLI1 is associated with 40 S ribosomal subunits in vivo, but it can interact with eIF3 and -5 independently of ribosomes. Depletion of RLI1 in vivo leads to cessation of growth, a lower polysome content, and decreased average polysome size. There was also a marked reduction in 40 S-bound eIF2 and eIF1, consistent with an important role for RLI1 in assembly of 43 S preinitiation complexes in vivo. Mutations of conserved residues in RLI1 expected to function in ATP hydrolysis were lethal. A mutation in the second ATP-binding cassette domain of RLI1 had a dominant negative phenotype, decreasing the rate of translation initiation in vivo, and the mutant protein inhibited translation of a luciferase mRNA reporter in wild-type cell extracts. These findings are consistent with a direct role for the ATP-binding cassettes of RLI1 in translation initiation. RLI1-depleted cells exhibit a deficit in free 60 S ribosomal subunits, and RLI1-green fluorescent protein was found in both the nucleus and cytoplasm of living cells. Thus, RLI1 may have dual functions in translation initiation and ribosome biogenesis.