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At least 19 recordsLinked to original sources
PubMed2005

High affinity RNA for mammalian initiation factor 4E interferes with mRNA-cap binding and inhibits translation.

The eukaryotic translation initiation factor 4F (eIF4F) consists of three polypeptides (eIF4A, eIF4G, and eIF4E) and is responsible for recruiting ribosomes to mRNA. eIF4E recognizes the mRNA 5'-cap structure (m7GpppN) and plays a pivotal role in control of translation initiation, which is the rate-limiting step in translation. Overexpression of eIF4E has a dramatic effect on cell growth and leads to oncogenic transformation. Therefore, an inhibitory agent to eIF4E, if any, might serve as a novel therapeutic against malignancies that are caused by aberrant translational control. Along these lines, we developed two RNA aptamers, aptamer 1 and aptamer 2, with high affinity for mammalian eIF4E by in vitro RNA selection-amplification. Aptamer 1 inhibits the cap binding to eIF4E more efficiently than the cap analog m7GpppN or aptamer 2. Consistently, aptamer 1 inhibits specifically cap-dependent in vitro translation while it does not inhibit cap-independent HCV IRES-directed translation initiation. The interaction between eIF4E and eIF4E-binding protein 1 (4E-BP1), however, was not inhibited by aptamer 1. Aptamer 1 is composed of 86 nucleotides, and the high affinity to eIF4E is affected by deletions at both termini. Moreover, relatively large areas in the aptamer 1 fold are protected by eIF4E as determined by ribonuclease footprinting. These findings indicate that aptamers can achieve high affinity to a specific target protein via global conformational recognition. The genetic mutation and affinity study of variant eIF4E proteins suggests that aptamer 1 binds to eIF4E adjacent to the entrance of the cap-binding slot and blocks the cap-binding pocket, thereby inhibiting translation initiation.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Chemical synthesis and binding activity of the trypanosomatid cap-4 structure.

Leishmania and other trypanosomatids are early eukaryotes that possess unusual molecular features, including polycistronic transcription and trans-splicing of pre-mRNAs. The spliced leader RNA (SL RNA) is joined to the 5' end of all mRNAs, thus donating a 5' cap that is characterized by complex modifications. In addition to the conserved m7GTP, linked via a 5'-5'-triphosphate bound to the first nucleoside of the mRNA, the trypanosomatid 5' cap includes 2'-O methylations on the first four ribose moieties and unique base methylations on the first adenine and the fourth uracil, resulting in the cap-4 structure, m7Gpppm3(6,6,2')Apm2'Apm2' Cpm2(3,2')U, as reported elsewhere previously. A library of analogs that mimic the cap structure to different degrees has been synthesized. Their differential affinities to the cap binding proteins make them attractive compounds for studying the molecular basis of cap recognition, and in turn, they may have potential therapeutic significance. The interactions between cap analogs and eIF4E, a cap-binding protein that plays a key role in initiation of translation, can be monitored by measuring intrinsic fluorescence quenching of the tryptophan residues. In the present communication we describe the multistep synthesis of the trypanosomatid cap-4 structure. The 5' terminal mRNA tetranucleotide fragment (pm3(6,6,2')Apm2'Apm2'Cpm2(3,2')U) was synthesized by the phosphoramidite solid phase method. After deprotection and purification, the 5'-phosphorylated tetranucleotide was chemically coupled with m7GDP to yield the cap-4 structure. Biological activity of this newly synthesized compound was confirmed in binding studies with eIF4E from Leishmania that we recently cloned (LeishIF4E-1), using the fluorescence time-synchronized titration method.

Animals↗
PubMed1984

mRNA discrimination in extracts from uninfected and reovirus-infected L-cells.

Uncapped reovirus mRNA extracted at late times from infected L-cells is preferentially translated in extracts from infected L-cells. However, translation of this uncapped, late, reovirus mRNA in extracts from infected cells is sensitive to inhibition by the cap analog m7GTP . These results imply that reovirus infection does not induce a transition from cap-dependent to cap-independent translation. Nevertheless, the results of in vitro translational competition experiments between L-cell mRNA and late viral mRNA were consistent with the view that reovirus does induce an alteration in the cap-dependent translational apparatus of L-cells. The reduced efficiency of translation of a variety of capped mRNAs in extracts from infected cells is also consistent with this notion. We further conclude that a factor exists in reovirus-infected L-cells that specifically stimulates translation of uncapped reovirus mRNAs.

Animals↗
PubMed1988

High-level synthesis in Escherichia coli of functional cap-binding eukaryotic initiation factor eIF-4E and affinity purification using a simplified cap-analog resin.

Numerous studies have established the important role that eukaryotic initiation factor-4E (eIF-4E) plays during protein biosynthesis. However, biochemical characterization of eIF-4E has proved difficult, mainly because of its low abundance in cells. To facilitate studies on eIF-4E, we have overexpressed Saccharomyces cerevisiae eIF-4E in Escherichia coli. The isolation of eIF-4E was simplified by using a cap-analog affinity matrix (agarose resin) that is considerably less demanding to prepare than those previously reported. We describe a simple and rapid purification scheme that can yield 2-5 micrograms of a homogenous and active preparation of eIF-4E from 1 ml of E. coli culture. E. coli-expressed eIF-4E is active as determined by its ability to bind the cap structure. The results demonstrate that the cap-binding activity of eIF-4E is not dependent on the presence of other proteins that are present at low levels in eIF-4E preparations isolated from eukaryotic cells.

Base Sequence↗
PubMed1994

Chromatographic resolution of in vivo phosphorylated and nonphosphorylated eukaryotic translation initiation factor eIF-4E: increased cap affinity of the phosphorylated form.

Eukaryotic translation initiation factor eIF-4E plays a central role in the recognition of the 7-methylguanosine-containing cap structure of mRNA and the formation of initiation complexes during protein synthesis. eIF-4E exists in both phosphorylated and nonphosphorylated forms, and the primary site of phosphorylation has been identified. Previous studies have suggested that eIF-4E phosphorylation facilitates its participation in protein synthesis. However, the biochemical basis for the functional difference between the two forms of eIF-4E is unknown. To address this directly, we have developed a method for the separation of phosphorylated and nonphosphorylated eIF-4E from rabbit reticulocytes by chromatography on rRNA-Sepharose. Using the resultant purified forms, we have studied the protein's interaction with the cap analogs m7GTP and m7GpppG and with the cap of globin mRNA by fluorescence quenching of tryptophan residues. It was found that phosphorylated eIF-4E had 3- to 4-fold greater affinity for cap analogs and mRNA than nonphosphorylated eIF-4E. The equilibrium binding constants (x 10(5), expressed as M-1) for the interaction of phosphorylated eIF-4E with m7GTP, m7GpppG, and globin mRNA were 20.0 +/- 0.1, 16.4 +/- 0.1, and 31.0 +/- 0.1, respectively, whereas those for the nonphosphorylated form were 5.5 +/- 0.4, 4.3 +/- 0.4, and 10.0 +/- 0.1, respectively. Treatment with potato acid phosphatase converted the phosphorylated form to the nonphosphorylated form and decreased the binding constant for m7GTP by a factor of 3. The increased affinity for mRNA caps may account for the in vivo and in vitro correlations between eIF-4E phosphorylation and accelerated protein synthesis and cell growth.

Animals↗
PubMed1982

Translational specificity in reovirus-infected mouse fibroblasts.

The specificity of protein synthesis in reovirus-infected mouse SC-1 cells was investigated, with the following results: a, extracts from infected and control cells translated saturating levels of capped globin mRNA with the same efficiency; b, infected cell extracts did not display a virus-induced increase in the capacity to translate decapped globin mRNA; c, translation of both cellular and viral messages was equally sensitive to the cap analog, 7-methylguanosine 5'-triphosphate. These findings are consistent with a previously published model (Walden, W., Godefroy-Colburn, T., and Thach, R. E. (1981) J. Biol. Chem. 256, 11739-11746) in which translation rates in reovirus-infected SC-1 cells are regulated by competition of capped host and viral mRNAs for a component of the unaltered protein synthetic system. Similar experimental results were obtained using reovirus-infected mouse L cells, with the following exceptions: a, the shutoff of host translation was far greater in L cells than in SC-1 cells; b, extracts of infected L cells were less active than controls for translation of all mRNAs tested. The reasons for the differences between SC-1 cells and L cells in their response to reovirus infection are being investigated.

Animals↗
PubMed1980

Detection of an enzyme activity which cleaves m7 guanine from m7 GMP in an extract of embryonic chick lens cells.

m7Guanine was cleaved from m7GMP by cytoplasmic enzyme activity in an extract prepared from embryonic chick lens cells. The appearance of m7-Guanine was proportional to the time and concentration of extract. m7-Guanine inhibited the reaction but neither guanine nor ribose 5-phosphate did. m7Guanine was not released from m7Guanosine. m7Guanine may be derived from m7GpppG mRNA cap by two enzymatic reactions with m7CMP as a product-substrate intermediate.

Animals↗
PubMed1981

Studies on the heterogeneity of the 5' ends of the protamine mRNAs from rainbow trout testis.

The structures of the 5' termini of the protamine mRNAs (PmRNAs) have been investigated by inhibiting their translation in wheat-germ extracts in the presence of 7-methyl guanosine 5'-phosphate (m7-GMP), an analogue of 'cap' structure in mRNAs. Second, the cap structures on PmRNAs were examined by labelling the RNA at the 5' end with T4 polynucleotide kinase and [gamma-32P]ATP before and after removal of these structures with tobacco acid pyrophosphatase and alkaline phosphatase. The results indicate that cap structures of the PmRNAs are heterogeneous. It appears that the mRNAs coding for protamine components CI and CIII have at least a cap 1 structure while the mRNAs coding for CII do not appear to be capped or methylated.

Animals↗
PubMed1984

Effect of 7-methylguanosine-5'-phosphate on the translation of rat milk-protein mRNAs in the wheat-germ cell-free system.

The translation of polyadenylated and of non-polyadenylated RNA obtained from lactating rat mammary gland was almost totally inhibited by 0.5 mM 7-methylguanosine-5'-phosphate in the wheat-germ cell-free system. This inhibition was maintained during the preparation of the 9S whey-protein mRNA and of the 12S and 15S casein mRNAs. Chemical decapping of these mRNAs caused a similar reduction of their activity. Although a large fraction of milk-protein mRNAs have been reported to lack 3'-polyadenylation, these results show that the mRNAs in the mammary gland do contain a 5'-terminal 7-methylguanosine cap.

Animals↗
PubMed1985

Detection of human autoantibodies specific for 5'-m7GMP and m7G(5')ppp(5')N.

An enzyme-linked immunosorbent assay was utilized for the detection of spontaneously occurring antibodies with apparent specificities for m7G, 5'-m7GMP, and m7G(5')ppp(5')C. From the sera of 50 patients containing anti-nuclear antibodies, 48 (96%) possessed antibodies which bound to one or more immobilized nucleoside-BSA antigens (A-, G-, C-, U-, and T-BSA). Additionally, 8 (16%) of these sera contained immunoglobulins that reacted with m7G-BSA antigen. In these latter sera, soluble competitors such as m7G, 5'm7GMP, and m7G(5')ppp(5')C (but not 5'-AMP, -GMP, -CMP, -UMP, and -TMP or m1G and m22G) effectively inhibited antibody-binding to immobilized m7G-BSA. These results indicate the existence of spontaneously occurring anti-m7G antibodies in autoimmune diseases which are distinct from anti-G antibody populations.

Antibodies, Antinuclear↗
PubMed1987

Reversion of 7-methylguanosine 5'-phosphate inhibition of mRNA translation by polysomal and soluble factors isolated from Saccharomyces cerevisiae.

Protein fractions that overcome m7GMP inhibition of mRNA translation have been purified from the yeast S. cerevisiae. An active fraction isolated from polysomes contains two polypeptides of 220- and 190-kDa. The active fraction isolated from postribosomal supernatant contains a major polypeptide of 28-kDa and other species of 32-, 24-, 22- and 21-kDa, and sediments in sucrose gradients as a high molecular weight complex of about 200,000. This fraction restored yeast mRNA translation in reticulocyte lysates under conditions of yeast and globin mRNA competition; however, this effect was not observed with the 220- and 190-kDa polypeptides from polysomes. Nevertheless, translation of yeast mRNA was stimulated by a partially purified fraction containing a 28-kDa polypeptide from polysomes.

Fungal Proteins↗