Isolation and sequence of a cDNA encoding the cap binding protein of wheat eukaryotic protein synthesis initiation factor 4F.
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Amino Acid Sequence↗
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Results for “RNA Cap-Binding Proteins”
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The mRNA 5' cap-binding protein, eIF-4E, cooperates with v-myc or E1A in the transformation of primary rodent fibroblasts.
We present evidence that eIF-4E, the mRNA 5' cap-binding protein, cooperates with two immortalizing oncogenes, v-myc and E1A, to cause transformation of rat embryo fibroblasts. eIF-4E alone can transform rat embryo fibroblasts when selection is applied. The pattern of transformation by eIF-4E is similar to that of p21 Ras, raising the possibility that eIF-4E shares a common signal transduction pathway with p21 Ras.
Adenovirus Early Proteins↗
A cap binding protein that may mediate nuclear export of RNA polymerase II-transcribed RNAs.
It has previously been shown that efficient export of U1 snRNA or of microinjected, in vitro synthesized, RNA transcripts from the nucleus of Xenopus oocytes is facilitated by their monomethyl guanosine cap structures. Nuclear exit of these transcripts could be competitively inhibited by microinjection of an excess of a cap analog, the dinucleotide m7GpppG (Hamm, J., and I. W. Mattaj. 1990. Cell. 63:109-118). We have now analyzed the ability of several other related cap analogs to inhibit the export of U1 snRNA from the nucleus. The results define the recognition specificity of a factor(s) involved in RNA transport, and indicate that the cap binding activity (CBA) involved in RNA export is different from cap binding proteins (CBPs) involved in the initiation of translation. A CBP, whose specificity for different analogs correlates with the ability of the analogs to inhibit U1 snRNA export, is identified in nuclear extracts prepared from HeLa cells. We propose that this protein may have a role in the export of capped RNAs from the nucleus.
Biological Transport, Active↗
GCR3 encodes an acidic protein that is required for expression of glycolytic genes in Saccharomyces cerevisiae.
Screening of a mutagenized strain carrying a multicopy ENO1-'lacZ fusion plasmid revealed a new mutation affecting several glycolytic enzyme activities. The recessive single nuclear gene mutation, named gcr3, caused an extremely defective growth phenotype on fermentable carbon sources such as glucose, while growth on respiratory media was almost normal. The GCR3 gene was obtained by growth complementation from a genomic DNA library, and the complemented strains had normal enzyme levels. GCR3 gene was sequenced, and a 99,537-Da protein was predicted. The predicted GCR3 protein was fairly acidic (net charge, -34). The C-terminal region was highly charged, and an acidic stretch was found in it.
Amino Acid Sequence↗
Selective in vivo and in vitro incorporation and accumulation of phenolic thioether amine into malignant melanoma and identification of a (58 kD) binding glycoprotein.
Our previous in vivo studies indicated that a phenolic thioether amine (PTEA), 4-S-cysteaminylphenol (CAP), selectively disintegrates melanocytes of black hair and skin, and inhibits the growth of murine and human malignant melanomas. To elucidate the mechanism of the in vivo melanocytotoxicity and anti-melanoma effect, this study examined the selectivity and specificity of PTEA incorporation into malignant melanoma cells using [14C]4(2)-S-CAP, and then identified a PTEA-binding protein through a ligand binding assay using [125I]-labelled cell lysates. Whole body autoradiography showed that [14C]4-S-CAP is selectively incorporated and accumulated into the eye and tumours of a B16 melanoma-bearing mouse. SK MEL 23 human melanoma cells also showed a steady accumulation of [14C]4-S-CAP (threefold at least up to 5 min) and of [14C]2-S-CAP (sevenfold up to 20 min), compared with that of HeLa cells and fibroblasts, which plateau at 5 min. Chromatography of 4-S-CAP on an affinity column (both CH- and CNBr-activated Sepharose 4B) identified a 58 kD protein in melanoma cells, which was present at very low levels in HeLa cells; this 58 kD protein was retained by both 4-S- and 2-S-CAP affinity columns, but not by columns of a phenolic thioether (cysteinylphenol: CP) or a phenolic thioether amide (N-acetyl-4-S-CAP), and could be retrieved by either 4-S or 2-S-CAP but not by CP and N-acetyl-4-S-CAP. This protein was glycosylated, and contained mannose residues.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Signal transduction and regulation of translation initiation.
Regulation of the rate of protein synthesis is important in the control of cellular proliferation. Changes in the rate of protein translation are brought about primarily at the level of initiation, which is usually rate limiting. This regulation involves the reversible phosphorylation of key initiation factors. Translation initiation factors eIF-4F, eIF-4B, and ribosomal protein S6 are phosphorylated in response to a wide variety of mitogens, growth factors, and tyrosine kinase oncogenes. Thus, translation initiation factors are important components of signal transduction pathways activated by extracellular factors and oncogenes. Of particular interest is the messenger RNA 5' cap-binding protein, eIF-4E. Overexpression of eIF-4E in fibroblasts results in malignant transformation, suggesting that it is an important transducer of growth signals, and that aberrant expression of a translation factor can cause malignancy. Elucidation of the components of the signalling pathways which regulate initiation factor activity should increase our understanding of how extracellular factors and oncogenes effect cellular proliferation, and the role that translation plays in this process.
Animals↗
Inhibition of the protein kinase PKR by the internal ribosome entry site of hepatitis C virus genomic RNA.
Translation of the hepatitis C genome is mediated by internal ribosome entry on the structurally complex 5' untranslated region of the large viral RNA. Initiation of protein synthesis by this mechanism is independent of the cap-binding factor eIF4E, but activity of the initiator Met-tRNA(f)-binding factor eIF2 is still required. HCV protein synthesis is thus potentially sensitive to the inhibition of eIF2 activity that can result from the phosphorylation of the latter by the interferon-inducible, double-stranded RNA-activated protein kinase PKR. Two virally encoded proteins, NS5A and E2, have been shown to reduce this inhibitory effect of PKR by impairing the activation of the kinase. Here we present evidence for a third viral strategy for PKR inhibition. A region of the viral RNA comprising part of the internal ribosome entry site (IRES) is able to bind to PKR in competition with double-stranded RNA and can prevent autophosphorylation and activation of the kinase in vitro. The HCV IRES itself has no PKR-activating ability. Consistent with these findings, cotransfection experiments employing a bicistronic reporter construct and wild-type PKR indicate that expression of the protein kinase is less inhibitory towards HCV IRES-driven protein synthesis than towards cap-dependent protein synthesis. These data suggest a dual function for the viral IRES, with both a structural role in promoting initiation complex formation and a regulatory role in preventing inhibition of initiation by PKR.
Animals↗
Dual initiation sites of protein synthesis on foot-and-mouth disease virus RNA are selected following internal entry and scanning of ribosomes in vivo.
The initiation of protein synthesis on foot-and-mouth disease virus RNA occurs at two sites separated by 84 nucleotides. Immediately upstream from the first of these sites is the internal ribosome entry site (IRES), which directs the translation of this RNA to be cap-independent. The utilization of these two initiation sites has been examined using artificial fusion genes in vivo under a variety of conditions. Additional in-frame AUG codons have been introduced between these two authentic start sites to determine the mechanism by which ribosomes recognize the second start site. The results indicate that following internal entry of ribosomes on the 5' side of the first initiation codon, many fail to initiate protein synthesis at this position and scan along the RNA to the second initiation site. In the presence or absence of the IRES both initiation sites are efficiently used but the utilization of the two sites is slightly biased towards the second initiation site by the IRES. Furthermore, in the presence of the IRES, protein synthesis initiates at both sites independently of the activity of the cap-binding complex.
Aphthovirus↗
Eukaryotic mRNA cap binding protein: purification by affinity chromatography on sepharose-coupled m7GDP.
A 24,000-dalton polypeptide that binds strongly and can be specifically crosslinked to the 5'-terminal cap structure m7GpppN in eukaryotic mRNAs has been detected in protein synthesis initiation factor preparations [Proc. Natl. Acad. Sci. USA (1978) 75, 4843--4847]. This polypeptide has been purified to apparent homogeneity by one chromatographic passage through an affinity resin prepared by coupling the levulinic acid O2',3'-acetal of m7GDP to AH-Sepharose 4B. Translation, in HeLa cell extracts, of capped mRNAs including Sindbis virus, reovirus, and rabbit globin mRNAs was stimulated by the cap-binding protein under conditions that did not increase translation of noncapped RNAs of encephalomyocarditis virus and satellite tobacco necrosis virus.
Animals↗
Nucleotide sequence changes in the polymerase basic protein 2 gene of temperature-sensitive mutants of influenza A virus.
Influenza A viruses bearing temperature-sensitive (ts) mutations are restricted in replication in the respiratory tract of animals and humans and are therefore attenuated. Nucleotide sequences were determined for the RNA segment coding for the polymerase basic protein 2 (PB2) from a panel of 12 influenza A/Udorn/307/72 (H3N2) ts viruses, previously characterized to have a ts mutation in the PB2 gene. Each of the viruses with a ts mutation in the PB2 gene had a single amino acid change located at position 65, 100, 112, 174, 298, 310, 386, 391, 556, or 658 of the PB2 protein. The sites of the single mutations were scattered throughout the length of the protein and occurred in regions that are highly conserved among the influenza A virus PB2 predicted amino acid sequences. Interestingly, the substitution of aspartic acid for asparagine at position 556 was found to lie within a region that has homology with cap-binding motifs of human and yeast proteins. Taken together, the findings of lesion sites in the A/Udorn/307/72 PB2 gene and the three reported amino acid changes at positions 265, 417, and 512 for A/AA/6/60, A/WSN/33, and A/FPV/Ros/34 ts PB2 genes, respectively, indicate that the PB2 gene can sustain a viable ts mutation at different sites. This information will allow us to construct cloned cDNA copies of the A/Udorn/307/72 PB2 gene mutagenized at specific sites. Different configurations of two or more ts mutations may be incorporated into the cDNA PB2 gene constructs. We have a host-range reassortant virus that should permit rescue of in vitro-produced transcripts of the PB2 gene into infectious virus. The rescue of these mutated PB2 RNA segments into an infectious influenza A virus may lead to the development of live attenuated reassortant virus vaccines that are satisfactorily attenuated, genetically stable, and immunogenic in humans.
Amino Acid Sequence↗
Isolation and sequence of the cDNAs encoding the subunits of the isozyme form of wheat protein synthesis initiation factor 4F.
The nucleotide sequences of the cDNAs for the two subunits, p82 and p28, of the isozyme form of wheat germ eukaryotic initiation factor 4F (eIF-(iso)4F) were determined. The cDNA for the p82 subunit encodes a polypeptide of 86,514 Da. The deduced amino acid sequence of p82 contains possible motifs for ATP binding, metal binding, and phosphorylation. The cDNA sequence for the small subunit, p28, which is a m7G cap-binding protein, encodes a polypeptide of 23,524 Da. The deduced amino acid sequence of p28 is similar (approximately 38%) to cap-binding proteins from yeast and mammals. The p28 of wheat eIF-(iso)4F does not contain a serine or threonine in the vicinity of the serine (Ser53) of mammalian cap-binding protein which is phosphorylated and shown to affect activity in mammalian cells.
Amino Acid Sequence↗
Lipopolysaccharide stimulates phosphorylation of eukaryotic initiation factor-4F in macrophages and tumor necrosis factor participates in this event.
Bacterial lipopolysaccharide (LPS) produces rapid changes in macrophage protein synthesis and function. Phosphorylation of the 25 kDa mRNA cap-binding protein (eIF-4E) in model systems regulates the efficiency of protein synthesis. We report that both LPS and tumor necrosis factor-alpha (TNF-alpha) stimulate phosphorylation of eIF-4E and the p220 component of eIF-4F in bone marrow-derived macrophages. Moreover, anti-TNF-alpha antibodies inhibit LPS-stimulated phosphorylation of eIF-4E and p220 by 43% (+/- 6%) and 50% (+/- 5%), respectively. Our results indicate that LPS stimulates eIF-4F phosphorylation by a TNF-alpha-dependent mechanism, and suggest that phosphorylation of eIF-4F might play a role in the post-transcriptional regulation of gene expression in macrophages exposed to LPS.
Animals↗
mRNAs containing extensive secondary structure in their 5' non-coding region translate efficiently in cells overexpressing initiation factor eIF-4E.
Cellular eukaryotic mRNAs (except organellar) contain at the 5' terminus the structure m7(5')Gppp(5')N (where N is any nucleotide), termed cap. Cap recognition by eukaryotic initiation factor eIF-4F plays an important role in regulating the overall rate of translation. eIF-4F is believed to mediate the melting of mRNA 5' end secondary structure and facilitate 43S ribosome binding to capped mRNAs. eIF-4E, the cap-binding subunit of eIF-4F, plays an important role in cell growth; its overexpression results in malignant transformation of rodent cells, and its phosphorylation is implicated in signal transduction pathways of mitogens and growth factors. The molecular mechanism by which eIF-4E transforms cells is not known. Here, we report that overexpression of eIF-4E facilitates the translation of mRNAs containing excessive secondary structure in their 5' non-coding region. This effect may represent one mechanism by which eIF-4E regulates cell growth and transforms cells in culture.
3T3 Cells↗
Positive regulation of the expression of the Escherichia coli pts operon. Identification of the regulatory regions.
The pts operon of Escherichia coli is composed of the ptsH, ptsI and crr genes coding for three proteins central to the phosphoenolpyruvate dependent phosphotransferase system (PTS), the HPr, enzyme I and EIIIGlc proteins, respectively. We previously showed that transcription from the promoter region located upstream from the pts operon is regulated by two control circuits, which can occur independently from each other. Transcription of the pts operon is (1) stimulated by the CAP-cAMP complex and (2) enhanced during growth on glucose, a PTS substrate. The DNA regions involved in regulation of the expression of the pts operon have been identified. Two promoters, P0 and P1, separated by 100 bp are located upstream from the pts operon. In these promoter regions, we identified two sequences showing similarity with the consensus of CAP-binding sites, CAPa located near P0 and CAPb located in the -35 region of P1. In vivo experiments showed that binding of CAP-cAMP at the CAPa site stimulates transcription from the P0 promoter. The binding sites of CAP-cAMP and/or RNA-polymerase on a DNA fragment containing both P0 and P1 promoters as well as both CAPa and CAPb sites were examined by the technique of DNase I footprinting. These in vitro experiments suggested that CAP-cAMP binding at the CAPb site might also play a role in regulation of the pts operon expression. In addition, we showed that the DNA region carrying the CAPa site is important for regulation by glucose. We finally propose that the expression of the pts operon is controlled by two alternative positive regulatory mechanisms, which are designed to allow activation of the pts operon under a great variety of growth conditions.
Amino Acid Sequence↗
A polypeptide in eukaryotic initiation factors that crosslinks specifically to the 5'-terminal cap in mRNA.
Protein synthesis initiation factors prepared from rabbit reticulocyte and mouse ascites ribosomes were tested for the ability to crosslink to the 5' cap of mRNA. Crosslinking of one polypeptide of apparent molecular weight 24,000 was inhibited by the cap analogs, m7GMP and m7GDP, indicating a specific interaction with the cap. Although specific crosslinking of the 24,000 molecular weight polypeptide was found with eukaryotic initiation factor 3 and to a lesser extent with initiation factor 4B, both of these factors contained less than stoichiometric amounts of this polypeptide. The crosslinking method provides a highly sensitive and specific assay for cap-binding proteins and should facilitate their purification for functional studies.
Chromatography, DEAE-Cellulose↗
A fraction of the mRNA 5' cap-binding protein, eukaryotic initiation factor 4E, localizes to the nucleus.
The 5' cap structure m7GpppN (where N is any nucleotide) is a ubiquitous feature of cellular eukaryotic mRNAs. The cap is multifunctional as it is involved in translation, nucleocytoplasmic transport, splicing, and stabilization of mRNA against 5' exonucleolytic degradation. The cap binding protein, eukaryotic initiation factor 4E (eIF-4E), is a translation initiation factor that binds to the cap structure and is part of a complex (eIF-4F) that promotes mRNA binding to ribosomes. Overexpression of eIF-4E in fibroblasts results in cell transformation. To test the hypothesis that some of the biological effects of eIF-4E might be effected by a nuclear function, we determined the cellular distribution of eIF-4E. By means of indirect immunofluorescence experiments using polyclonal and monoclonal antibodies against eIF-4E as well as transfected epitope-tagged eIF-4E, we demonstrate that a fraction of eIF-4E localizes to the nucleus. These results suggest that eIF-4E is also involved in a nuclear function.
Amino Acid Sequence↗
Interaction of initiation factors with the cap structure of chimaeric mRNA containing the 5'-untranslated regions of Semliki Forest virus RNA is related to translational efficiency.
Chimaeric chloramphenicol acetyltransferase (CAT) mRNA, containing the leader sequences of genomic 42S RNA and subgenomic 26S RNA of Semliki Forest virus (SFV) were synthesized by in-vitro transcription. These transcripts were translated with different efficiencies, as the authentic mRNA in SFV-infected cells. Therefore, they can be used as model mRNA species to study the mechanism underlying SFV-directed shut off of host protein synthesis. The interaction of translation initiation factors with the 5' cap structure was studied. Transcripts prepared in vitro using T7 RNA polymerase were capped and methylated posttranscriptionally with [32P]-GTP and S-adenosyl-L-methionine to yield cap-labelled mRNA species. Irradiation with ultraviolet light of 26S CAT and 42S CAT transcripts, together with crude rabbit reticulocyte initiation factors, resulted in the cap-specific cross-linking of eukaryotic initiation factors (eIF) eIF-4E and eIF-4B. The relative binding efficiency of these two factors to the cap structure of the various transcripts was, however, markedly different; the cap structure present in 26S CAT mRNA interacted efficiently with cap-binding proteins, whereas the cap structure of 42S CAT mRNA hardly bound to these proteins. Comparable results were obtained under competitive conditions. Data are presented that the secondary structure close to the 5' cap structure determines the efficiency of recognition of the mRNA by these initiation factors. Using a chemical cross-linking assay, it was demonstrated that eIF-4F, and also eIF-4E, differentially interacted with the cap structure of the various transcripts. The data are discussed with respect to the possible mechanisms involved in SFV-induced shut off of host cell protein synthesis.
Base Sequence↗
Positive autoregulation of the Vibrio fischeri luxR gene. LuxR and autoinducer activate cAMP-catabolite gene activator protein complex-independent and -dependent luxR transcription.
The LuxR protein is a transcriptional activator involved in regulation of the genes required for bioluminescence (lux) in the marine bacterium Vibrio fischeri. Transcription of the two divergently oriented lux operons (luxR and luxICDABEG) is activated by LuxR in the presence of a diffusible inducer (autoinducer). Transcription of the luxR gene is subject to both positive and negative autoregulation as well as activation by the cAMP-catabolite gene activator protein complex (cAMP-CAP). Transcription of luxR was studied using both luminescence in vivo as a reporter and primer extension analysis of mRNA synthesized in vivo. Mutation of the lux CAP-binding site resulted in a reduction in luminescence from the reporter and the complete loss of luxR positive autoregulation. Positive autoregulation was restored if luxR was provided in trans, demonstrating that LuxR and autoinducer activate luxR transcription in the absence of cAMP-CAP. By means of primer extension analysis, three sites of initiation of luxR transcription were demonstrated; initiation at two of these sites required cAMP-CAP. The quantity of all three transcripts was increased in the presence of LuxR and autoinducer when a plasmid with a wild-type CAP-binding site was used. Initiation at the cAMP-CAP-dependent sites was not observed from a plasmid with a mutated CAP-binding site in the presence or absence of autoinducer even with luxR supplied in trans. Instead, with luxR supplied in trans, initiation at the cAMP-CAP-independent initiation site was specifically stimulated by LuxR and autoinducer. Thus, in the course of positive autoregulation, the LuxR protein activates transcription from two luxR promoters by a cAMP-CAP-dependent mechanism and a third promoter by a cAMP-CAP-independent mechanism.
Bacterial Proteins↗