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PubMed1989

The specific guanine binding site of the ribonuclease T1 family enzymes and of G-proteins is modeled in the cocrystal formed by 7-methylguanosine-5'-phosphate and phenylalanine.

In the cocrystal formed by 7-methylguanosine-5'-phosphate.phenylalanine.6H2O, the interactions between guanine and phenylalanine are similar to those observed in the complex of ribonuclease T1 with 2'-guanylic acids, and those of the two G-proteins, Elongation Factor-Tu and ras oncogene p21, with GDP. They are similar in the following three points: (a) guanine N(1)H and N(2)H donate cyclic N-H...O hydrogen bonds to the carboxylate group of phenylalanine in the former cocrystal and to the side chain carboxylate group of Asp or Glu in the latter proteins, (b) O(6) of guanine accepts hydrogen bond(s) from main-chain NH group(s), and (c) the purine moiety is sandwiched between aromatic (or hydrophobic) amino acid side chains.

Binding Sites↗
PubMed1981

Action of nucleotide derivatives on translation in encephalomyocarditis virus-infected mouse cells.

The infection of animal cells by encephalomyocarditis (EMC) virus lead to a drastic change in membrane permeability towards low mol. wt. compounds. Addition of the nucleotide analogue GppCH2p to the culture medium resulted in a specific inhibition of protein synthesis in EMC virus-infected 3T6 cells. This inhibition was not observed when GTP or ATP were present nor in control mock-infected 3T6 cells. The induction of membrane leakiness after viral infection was not specific for 3T6 cells, as it was also detected in mouse L cells, hamster BHK-21 cells and monkey CV1 cells. The inhibitory action produced by GppCH2p in virus-infected cells was fully reversed upon addition of fresh medium. Moreover, analysis of the proteins synthesized after medium replacement showed a preferential synthesis of cellular proteins. The presence of zinc ions resulted in an inhibition of the cleavage of large viral polypeptide precursors to mature viral proteins. Under these conditions, membrane leakiness as measured by GppCH2p, was not observed. However, this seems to be an effect of zinc ions themselves on the membrane, because inhibition of mature protein formation by other means, such as the presence of amino acid analogues, did not prevent inhibition of translation by GppCH2p in virus-infected cells. Addition of the cap analogues 7mGppp and 2'-O'-mGppp, resulted in specific stimulation of viral protein synthesis in EMC virus-infected 3T6 cells. On the other hand, the presence of 7mGp had no effect on translation. We propose that a specific capping of viral mRNA takes place in the presence of these compounds, and leads to increased stability and greater efficiency in the translation of viral mRNA.

Diphosphonates↗
PubMed1989

Effect of the cap structure on pre-mRNA splicing in Xenopus oocyte nuclei.

The effect of the 5' cap structure on the splicing of precursor mRNAs was investigated after the RNAs were injected into Xenopus oocyte nuclei. The precursor mRNAs synthesized in vitro in a prokaryotic transcription system with a dinucleotide, ApppG, as a primer, were extremely stable when injected into the nuclei yet behaved like uncapped pre-mRNAs in the in vitro splicing reaction. The ApppG-primed precursor mRNAs served as a control (uncapped) in the injection experiments, and their splicing reactions were compared with those of their capped (m7GpppG-primed) counterparts. The capped precursors were spliced more efficiently than the uncapped precursors. Examination of splicing of the precursor mRNA that contained three exons and two introns with a single molecule has revealed that the cap structure exerts its effect primarily on the 5'-proximal intron. Thus, the cap structure not only stabilizes precursor mRNAs but also plays a positive role in the splicing of precursor mRNAs in cells.

Animals↗
PubMed1980

Inhibitory effects of 'cap' analogues on globin mRNA and encephalomyocarditis RNA translation in a reticulocyte cell-free system.

The cap analogues 7-methylguanosine 5'-phosphate [m7G(5')p], 7-methylguanosine 5'-triphosphate [m7G(5')ppp] and 2'-O-methylguanosine 5'-triphosphate [Gm(5')ppp] inhibit the translation of capped globin mRNA and encephalomyocarditis (EMC) RNA (a naturally uncapped mRNA) in a reticulocyte cell-free system. This inhibition occurs at the level of protein synthesis initiation and is of a competitive type since it can be overcome by increasing the mRNA concentration. However, the translation of globin mRNA is more sensitive to the inhibitory effects of the cap analogues m7G(5')p and m7G(5')ppp than translation of EMC RNA. The same spectra of specific inhibition is also observed with some other initiation inhibitors such as aurintricarboxylic acid, which inhibits mRNA binding, but not with pactamycin which does not affect mRNA interaction. A model is presented suggesting that this preferential inhibition by cap analogues could be explained mainly by the different affinities of globin mRNA and EMC RNA for the initiation complexes between 40-S subunits and Met-tRNAf. Moreover Gm(5')ppp cannot be considered simply as a cap analogue since it also affects some step prior to mRNA binding.

Animals↗
PubMed1996

Analysis of the mRNA cap-binding ability of human eukaryotic initiation factor-4E by use of recombinant wild-type and mutant forms.

In order to identify the amino acid residues necessary for the selective recognition of the mRNA cap structure by human eukaryotic initiation factor-4E (eIF-4E), which plays a central role in the first step of mRNA translation, we prepared recombinant wild-type and fourteen mutant forms and compared their cap-binding abilities by affinity chromatography. By the direct expression of a synthetic gene encoding human eIF-4E as the soluble form in Escherichia coli and the application on a 7-methylguanosine-5'-triphosphate-Sepharose 4B cap affinity column, pure recombinant eIF-4E was prepared; the optimum pH for the binding of the mRNA cap was 7.5. Among the amino acid residues conserved among various eIF-4E species, each of 14 functional residues was replaced with a nonpolar amino acid (alanine or leucine). All mutant eIF-4E genes, which were constructed by site-directed mutagenesis, were expressed in the same way as the wild type, and their cap-binding abilities were compared with that of the wild type. Consequently, all eight tryptophan residues. Glu103, and two histidine residues at positions 37 and 200 in human recombinant eIF-4E were suggested to be important for the recognition of the mRNA cap structure through direct interaction and/or indirect contributions. Indirect contributions included the construction of the overall protein structure, especially the cap-binding pocket.

Amino Acid Sequence↗
PubMed1982

Effects of cap analogue or cap removal on the translation of rat brain mRNA in vitro.

The role of cap structures in the translation of brain mRNA was examined by measuring protein biosynthesis in vitro in wheat germ and reticulocyte systems programmed by mRNA that was either untreated or oxidized by periodate or from which 5'-terminal 7-methylguanosine (m7G) was removed by oxidation and beta-elimination. In another series of reactions, amino acid incorporation into polypeptides was measured in the absence and in the presence of varying concentrations of the cap analogue 7-methylguanosine 5'-triphosphate (pppm7G). The results indicated that any of the above treatments interfered with brain mRNA translation, the degree of inhibition depending on the translation system used, the concentration of mRNA, and the source of initiation factors. Homologous brain initiation factors were superior to reticulocyte factors in providing a partial relief from inhibition of translation caused by these treatments. It was also found that synthesis of the brain-specific protein S-100 was inhibited by beta-elimination of mRNA, by pppm7G, or by the presence of capped globin mRNA, indicating that the mRNA for this protein was probably capped.

Animals↗
PubMed1990

Fluorometric analysis of the long-wavelength absorption band of N-7 methylated GMP into the constituent bands of the two electronic states.

Steady-state measurements of fluorescence anisotropy are used to resolve the long-wavelength absorption spectrum of 7-methyl guanosine 5'-monophosphate (GMP) in pH 5 buffer at room temperature into component spectra that correspond to the electronic transitions I and II present in that spectral region. We have chosen this derivative of guanine because its fluorescence quantum yield is much greater than that of GMP. It is found that the data are adequately described by a model that involves emission exclusively from state I, with state II converting to it with 100% efficiency. The shape of the absorption spectrum of state II is virtually independent of the angle theta between the absorption transition dipole moments of states I and II, whereas that of state I is dependent on theta. We analyze the data on the basis of the premise that in the short-wavelength region state II is the predominantly absorbing state. This premise is based on studies of single-crystal polarized reflection and linear dichroism from stretched films. The spectral maxima for the two states are found to be at about 290 and 260 nm, respectively. There is also a weak band which is centered at about 245 nm. The oscillator strengths are found to be 0.07, 0.21 and approximately 0.04, for states I, II and that associated with the weak band, respectively. The importance of these findings with regard to the photophysical properties of nucleic acids and calculations of their CD spectra is discussed.

Fluorescence Polarization↗
PubMed2006

Binding specificities and potential roles of isoforms of eukaryotic initiation factor 4E in Leishmania.

The 5' cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the Leishmania genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m(7)GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m(7)GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1, though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E; thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant in amastigotes and also contains a typical 3' untranslated region element that is found in amastigote-specific genes. LeishIF4E-2 bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3 bound mainly m(7)GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the Saccharomyces cerevisiae eIF4E, indicating that despite their structural conservation, they are considerably diverged.

3' Untranslated Regions↗
PubMed1997

Critical residues of Semliki Forest virus RNA capping enzyme involved in methyltransferase and guanylyltransferase-like activities.

The Semliki Forest virus (SFV) replicase protein nsP1 has methyltransferase (MT) and guanylyltransferase-like (GT) activities, which are involved in the capping of viral mRNAs. MT catalyzes the transfer of the methyl group from S-adenosylmethionine (AdoMet) to position 7 of GTP, and this reaction is followed by GT-catalyzed formation of the covalent complex m7GMP-nsP1. These reactions are virus specific and thus potential targets for inhibitors of virus replication. We have mutated residues of SFV nsP1, which are conserved in related proteins of the large alphavirus-like superfamily. Mutations of D64, D90, R93, C135, C142, and Y249 to alanine destroyed or greatly reduced the MT activity of nsP1. All MT-negative mutants lost also the GT activity, confirming that methylation of GTP is an essential prerequisite for the synthesis of the covalent guanylate complex. Mutation of H38 prevented the GT reaction without destroying MT activity. Conservation of residues essential for both reactions in the alphavirus-like superfamily implies that they use a capping mechanism similar to that for the alphaviruses. Residues D64 and D90 were necessary for AdoMet binding, as measured by UV cross-linking. Secondary structure predictions of nsP1 and other proteins of the superfamily place these residues in positions corresponding to AdoMet-binding sites of cellular methyltransferases, suggesting that they all may be structurally related.

Amino Acid Sequence↗
PubMed2001

Activity of the hepatitis A virus IRES requires association between the cap-binding translation initiation factor (eIF4E) and eIF4G.

The question of whether translation initiation factor eIF4E and the complete eIF4G polypeptide are required for initiation dependent on the IRES (internal ribosome entry site) of hepatitis A virus (HAV) has been examined using in vitro translation in standard and eIF4G-depleted rabbit reticulocyte lysates. In agreement with previous publications, the HAV IRES is unique among all picornavirus IRESs in that it was inhibited if translation initiation factor eIF4G was cleaved by foot-and-mouth disease L-proteases. In addition, the HAV IRES was inhibited by addition of eIF4E-binding protein 1, which binds tightly to eIF4E and sequesters it, thus preventing its association with eIF4G. The HAV IRES was also inhibited by addition of m(7)GpppG cap analogue, irrespective of whether the RNA tested was capped or not. Thus, initiation on the HAV IRES requires that eIF4E be associated with eIF4G and that the cap-binding pocket of eIF4E be empty and unoccupied. This suggests two alternative models: (i) initiation requires a direct interaction between an internal site in the IRES and eIF4E/4G, an interaction which involves the cap-binding pocket of eIF4E in addition to any direct eIF4G-RNA interactions; or (ii) it requires eIF4G in a particular conformation which can be attained only if eIF4E is bound to it, with the cap-binding pocket of the eIF4E unoccupied.

5' Untranslated Regions↗
PubMed2004

Critical residues for GTP methylation and formation of the covalent m7GMP-enzyme intermediate in the capping enzyme domain of bamboo mosaic virus.

Open reading frame 1 of Bamboo mosaic virus (BaMV), a Potexvirus in the alphavirus-like superfamily, encodes a 155-kDa replicase responsible for the formation of the 5' cap structure and replication of the viral RNA genome. The N-terminal domain of the viral replicase functions as an mRNA capping enzyme, which exhibits both GTP methyltransferase and S-adenosylmethionine (AdoMet)-dependent guanylyltransferase activities. We mutated each of the four conserved amino acids among the capping enzymes of members within alphavirus-like superfamily and a dozen of other residues to gain insight into the structure-function relationship of the viral enzyme. The mutant enzymes were purified and subsequently characterized. H68A, the mutant enzyme bearing a substitution at the conserved histidine residue, has an approximately 10-fold increase in GTP methyltransferase activity but completely loses the ability to form the covalent m(7)GMP-enzyme intermediate. High-pressure liquid chromatography analysis confirmed the production of m(7)GTP by the GTP methyltransferase activity of H68A. Furthermore, the produced m(7)GTP sustained the formation of the m(7)GMP-enzyme intermediate for the wild-type enzyme in the presence of S-adenosylhomocysteine (AdoHcy), suggesting that the previously observed AdoMet-dependent guanylation of the enzyme using GTP results from reactions of GTP methylation and subsequently guanylation of the enzyme using m(7)GTP. Mutations occurred at the other three conserved residues (D122, R125, and Y213), and H66 resulted in abolition of activities for both GTP methylation and formation of the covalent m(7)GMP-enzyme intermediate. Mutations of amino acids such as K121, C234, D310, W312, R316, K344, W406, and K409 decreased both activities by various degrees, and the extents of mutational effects follow similar trends. The affinity to AdoMet of the various BaMV capping enzymes, except H68A, was found in good correlations with not only the magnitude of GTP methyltransferase activity but also the capability of forming the m(7)GMP-enzyme intermediate. Taken together with the AdoHcy dependence of guanylation of the enzyme using m(7)GTP, a basic working mechanism, with the contents of critical roles played by the binding of AdoMet/AdoHcy, of the BaMV capping enzyme is proposed and discussed.

Amino Acid Sequence↗
PubMed1990

trans-spliced Caenorhabditis elegans mRNAs retain trimethylguanosine caps.

The nematode Caenorhabditis elegans has an unusual small nuclear RNA, containing a 100-nucleotide RNA molecule, spliced leader RNA, which donates its 5' 22 nucleotides to a variety of recipient RNAs by a trans-splicing reaction. The spliced leader RNA has a 5' trimethylguanosine (TMG) cap, which becomes the 5' end of trans-spliced mRNAs. We found that mature trans-spliced mRNAs were immunoprecipitable with anti-TMG cap antibodies and that TMG-containing dinucleotides specifically competed with the trans-spliced mRNAs for antibody binding. We also found that these mRNAs retained their TMG caps throughout development and that the TMG-capped mRNAs were polysome associated. Since the large majority of C. elegans mRNAs are not trans-spliced, the addition of the spliced leader and its TMG cap to a limited group of recipient RNAs may create a functionally distinct subset of mRNAs.

Actins↗
PubMed1990

mRNAs that mature through trans-splicing in Caenorhabditis elegans have a trimethylguanosine cap at their 5' termini.

Approximately 10% of the mRNAs in the nematode Caenorhabditis elegans mature through a trans-splicing mechanism that involves the transfer of a 22-nucleotide spliced leader to the 5' end of the pre-mRNA. The spliced leader RNA exists as a small nuclear ribonucleoprotein particle and has the trimethylguanosine cap that is characteristic of eucaryotic small nuclear RNAs. We found that the trimethylguanosine cap present on the spliced leader RNA was transferred to the pre-mRNA during the trans-splicing reaction. Thereafter, the trimethylguanosine cap was maintained on the mature mRNA. This is the first example of eucaryotic cellular mRNAs possessing a trimethylguanosine cap structure.

Animals↗
PubMed1997

Binding of eukaryotic translation initiation factor 4E (eIF4E) to eIF4G represses translation of uncapped mRNA.

mRNA translation in crude extracts from the yeast Saccharomyces cerevisiae is stimulated by the cap structure and the poly(A) tail through the binding of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) and the poly(A) tail-binding protein Pab1p. These proteins also bind to the translation initiation factor eIF4G and thereby link the mRNA to the general translational apparatus. In contrast, uncapped, poly(A)-deficient mRNA is translated poorly in yeast extracts, in part because of the absence of eIF4E and Pab1p binding sites on the mRNA. Here, we report that uncapped-mRNA translation is also repressed in yeast extracts due to the binding of eIF4E to eIF4G. Specifically, we find that mutations which weaken the eIF4E binding site on the yeast eIF4G proteins Tif4631p and Tif4632p lead to temperature-sensitive growth in vivo and the stimulation of uncapped-mRNA translation in vitro. A mutation in eIF4E which disturbs its ability to interact with eIF4G also leads to a stimulation of uncapped-mRNA translation in vitro. Finally, overexpression of eIF4E in vivo or the addition of excess eIF4E in vitro reverses these effects of the mutations. These data support the hypothesis that the eIF4G protein can efficiently stimulate translation of exogenous uncapped mRNA in extracts but is prevented from doing so as a result of its association with eIF4E. They also suggest that some mRNAs may be translationally regulated in vivo in response to the amount of free eIF4G in the cell.

Amino Acid Sequence↗
PubMed1988

Discontinuously synthesized mRNA from Trypanosoma brucei contains the highly methylated 5' cap structure, m7GpppA*A*C(2'-O)mU*A.

Trypanosoma brucei mRNA is discontinuously synthesized via the 5' addition of a "mini-exon" sequence. The mini-exon-specific cap structure was purified from a complete RNase T2 and phosphatase digest of in vivo 32P-labeled poly(A)+RNA. The purified cap structure was sequenced by a series of partial and complete enzymatic digests by nuclease P1 and venom phosphodiesterase. This approach demonstrated that the T. brucei mini-exon cap structure consists of N7-methylguanosine linked in a conventional 5'-5' triphosphate bond to five nucleotides, in the sequence A*A*C(2'-O)mU*A (asterisks denote modifications that were not fully characterized in this work). 2'-O-methylations and other modifications appear to be present in this novel cap structure, which could have a functional role in the metabolism of the mini-exon.

Animals↗
PubMed1988

Translational control in heat-shocked Drosophila embryos. Evidence for the inactivation of initiation factor(s) involved in the recognition of mRNA cap structure.

Lysates from normally growing (25 degrees C) or heat shocked (37 degrees C, 45 min) Drosophila melanogaster embryos were obtained and the effect of analogues of the mRNA 5'-terminal cap, m7G(5')ppp(5')N structure and of potassium ions on their endogenous protein synthesis activity was studied. At optimal concentration of KCH3COO (75-80 mM), protein synthesis in normal lysates is strongly inhibited by cap analogues (m7GpppG, m7GDP, and m7GMP). At the same ionic conditions, heat shock lysates translate preferentially the heat shock messengers, and this translation is almost unaffected by the cap analogues. In contrast, residual synthesis of normal proteins in heat shock lysates was reduced by these compounds. By lowering the concentration of potassium ions it was possible to gradually reverse the inhibitory effect of the cap analogues in normal lysates and also to increase specifically the translation of normal mRNAs in heat shock lysates. Translation of normal mRNAs is also partial but specifically rescued by supplementing heat shock lysates with polypeptide chain initiation factors partially purified from rabbit reticulocytes. These data are consistent with the notion that the failure of normal mRNAs to be translated under heat shock conditions might be due, at least to some extent, to the inactivation of polypeptide chain initiation factor(s) involved in the recognition of the mRNA 5'-terminal cap structure.

Animals↗
PubMed1984

Chemical carcinogenesis. II. Imidazole-ring-opened derivatives from 7-ethyl- and 1,7-diethylguanosine.

The UV-spectral and chromatographic analyses of the derivatives of the two synthetic standards 7-ethylguanosine and 1,7-diethylguanosine are here reported. The derivatives obtained from the dialkyl compound exhibit a striking similarity to those found in the "pyrimidine-nucleotide-like" fraction of rat liver tRNA ethylated in vivo by ethionine. The finding of imidazole-ring-opened products in tRNA ethylation by ethionine could be significant from the point of view of chemical carcinogenesis: in fact, imidazole-ring-opening of 1,7-dialkylguanosines directly at level of RNA with consequent formation of substituted pyrimidines is a transversion, i.e. a mutagenic event which would cause a change in the expression of genetic information since a purine has been transformed into a pyrimidine.

Animals↗