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PubMed1980

The addition of 5' cap structures occurs early in hnRNA synthesis and prematurely terminated molecules are capped.

After cells were labeled by brief exposure to 3H-methyl-L-methionine, the majority of labeled 5' terminal cap I (m7GpppN1mpN2p) oligonucleotide structures were in nuclear RNA (hnRNA) molecules approximately 750 nucleotides or less in length. After longer label times, the proportion of cap I structures in nuclear molecules longer than mRNA rose to approximately 60% of the total, but approximately 40% of the cap I structures were still in molecules shorter than approximately 750 nucleotides. The cap I structures in both long and short hnRNA chains contained all four 2' methylated nucleotides in the N1 position in about the same proportion as in mRNA. None of the large hnRNA molecules could be demonstrated to contain 5' pppX p termini; the only such terminus in high molecular weight RNA was pppAp which was decreased markedly by low doses of actinomycin and is presumably the terminus of pre-rRNA. These results raise the possibilities that hnRNA chains can initiate with any of the four nucleotides, that capping occurs very close to or at the start of hnRNA chain synthesis and that approximately 40% of the hnRNA chains may be prematurely terminated.

Animals↗

PubMed1984

Capped mRNAs may stimulate the influenza virion polymerase by allosteric modulation.

Analogues of the mRNA 5'-terminal methyl cap structure were found to stimulate the influenza virion RNA-dependent RNA polymerase. The single nucleotide analogue m7GMP was incorporated into RNA during transcription in vitro, and the stimulatory effect was not additive with the primer ApG, suggesting that m7GMP stimulates the virion polymerase by priming virus-specific mRNA synthesis, as has been shown for ApG. By contrast, stimulation by m7G(5')ppp(5')m6AM2-O was additive with that by ApG, and we could not demonstrate incorporation of the similar analogue m7G(5')ppp(5')Am2-O into RNA during transcription. We propose that these dinucleotide cap analogues stimulate the virion polymerase by allosteric modulation, independent of priming. This stimulation can be abolished by mutation, without loss of other activities associated with the cap-dependent endonuclease.

Allosteric Regulation↗

PubMed1992

Interaction of protein synthesis initiation factors with the mRNA cap structure.

The mechanism of mRNA recognition by proteins interacting with the mRNA cap structure was investigated by photochemical cross-linking of proteins with 32P-labelled reoviral RNAs. Using ribosomal washes as a source of eukaryotic protein synthesis initiation factors, we identified the well-known cap binding proteins eIF-4B and -4E, but eIF-2 and eIF-3 as well. The interplay of purified eIF-4A, -4B, and -4F was studied in relation to ATP dependence and cap analogue sensitivity of cap binding. Next to their well-known roles in the initiation process, eIF-2 and eIF-3 also cross-linked to the 5' cap. eIF-2 stimulated eIF-4B and -4E cross-linking, an observation that has been previously described more extensively. The interaction of eIF-2 with the 5' end of mRNA was extremely sensitive to K(+)-ions and was resistant to a high concentration of Mg(2+)-ions; this influence of mono- and divalent ions was in contrast with the cross-linking of eIF-4B and -4E. Optimal interaction of these factors was obtained at moderate K+ concentration and low Mg(2+)-ion concentrations. eIF-2 cross-linking was sensitive to high protein to mRNA ratios indicating a weak affinity as compared to eIF-4E and -4B. The interaction of eIF-3 with the cap of mRNA is also weak as it was counteracted by all other cap binding proteins, leading to an inability to detect the cross-linking of this protein in crude eIF preparations. Time kinetics of formation of complexes suggested eIF-2 to be one of the first factors to interact with mRNA. Preformed RNA-protein complexes were dissociated after cap analogue addition, suggesting reversible interactions between RNA and proteins.

Adenosine Triphosphate↗

PubMed1989

Base stacking of simple mRNA cap analogues. Association of 7,9-dimethylguanine, 7-methylguanosine and 7-methylguanosine 5'-monophosphate with indole and purine derivatives in aqueous solution.

Equilibrium constants for the association of different ionic forms of 7,9-dimethylguanine, 7-methylguanosine and 7-methylguanosine 5'-monophosphate with indole, caffeine and various methylated adenines have been determined by distributing the latter compounds between an organic solvent and aqueous solutions of the 7-methylguanine derivatives. The data are compared to those obtained for the association of unsubstituted purine with the same cosolutes. The stacking affinity of both cationic and zwitterionic forms of the 7-methylguanine ring correlates with the ring polarizability rather than the polarizing power of the cosolute. The cationic species stacks usually more efficiently. The chemical nature of the N9-substituent has only a moderate influence on the base-stacking properties.

Caffeine↗

PubMed2005

Crystal structures of human DcpS in ligand-free and m7GDP-bound forms suggest a dynamic mechanism for scavenger mRNA decapping.

Eukaryotic cells utilize DcpS, a scavenger decapping enzyme, to degrade the residual cap structure following 3'-5' mRNA decay, thereby preventing the premature decapping of the capped long mRNA and misincorporation of methylated nucleotides in nucleic acids. We report the structures of DcpS in ligand-free form and in a complex with m7GDP. apo-DcpS is a symmetric dimer, strikingly different from the asymmetric dimer observed in the structures of DcpS with bound cap analogues. In contrast, and similar to the m7GpppG-DcpS complex, DcpS with bound m7GDP is an asymmetric dimer in which the closed state appears to be the substrate-bound complex, whereas the open state mimics the product-bound complex. Comparisons of these structures revealed conformational changes of both the N-terminal swapped-dimeric domain and the cap-binding pocket upon cap binding. Moreover, Tyr273 in the cap-binding pocket displays remarkable conformational changes upon cap binding. Mutagenesis and biochemical analysis suggest that Tyr273 seems to play an important role in cap binding and product release. Examination of the crystallographic B-factors indicates that the N-terminal domain in apo-DcpS is inherently flexible, and in a dynamic state ready for substrate binding and product release.

Apoproteins↗

PubMed2003

Charge distribution in 7-methylguanine regarding cation-pi interaction with protein factor eIF4E.

Electric charge distribution in mRNA 5' cap terminus has been exhaustively characterized in respect to the affinity for cap-binding proteins. Formation of the stacked configuration of positively charged 7-methylguanine in between two aromatic amino acid rings, known as sandwich cation-pi stacking, is thought to be prerequisite for the specific recognition of the cap by eukaryotic initiation factor eIF4E; i.e., discrimination between the cap and nucleotides without the methyl group at N(7). Nuclear magnetic resonance spectroscopy of (15)N/(13)C-double-labeled 7-methylguanosine 5'-triphosphate and 7-methylguanosine, as well as their unsubstituted counterparts, GTP and guanosine, yielded characteristic changes of the electron-mediated spin-spin couplings and chemical shifts due to the methylation at N(7). The experimentally measured changes of the nuclear magnetic resonance parameters have been analyzed in respect to the electric charge distribution calculated by means of quantum chemical methods, and interpreted in terms of new proposed positive charge localization in the 7-methylguanine five-member ring.

Biophysical Phenomena↗

PubMed2002

Mutations in the S4-H2 loop of eIF4E which increase the affinity for m7GTP.

Eukaryotic initiation factor 4E (eIF4E) binds the 5'-cap of eukaryotic mRNAs and overexpression of eIF4E in epithelial cell cancers correlates with the metastases/tissue invasion phenotype. Photolabeling of eIF4E with [gamma-32P]8-azidoguanosine 5'-triphosphate (8-N3GTP) demonstrated cross-linking at Lys-119 in the S4-H2 loop which is distant from the m7GTP binding site [Marcotrigiano et al. (1997) Cell 89, 951-961; Friedland et al. (1997) Protein Sci. 6, 125-131]. Modeling studies indicate that 8-N3GTP cross-linked with Lys-119 because it binds a site that is occupied by the second nucleotide of a bound mRNA. Mutagenesis of the S4-H2 loop produced proteins with a 5-10-fold higher affinity for m7GTP than wild-type eIF4E. These mutants of eIF4E may have uses in selectively purifying mRNAs with intact 5'-ends or in determining how the promyelocytic leukemia protein decreases the affinity of eIF4E for mRNA caps.

Animals↗

PubMed2000

Interaction between a poly(A)-specific ribonuclease and the 5' cap influences mRNA deadenylation rates in vitro.

We have used an in vitro system that reproduces in vivo aspects of mRNA turnover to elucidate mechanisms of deadenylation. DAN, the major enzyme responsible for poly(A) tail shortening in vitro, specifically interacts with the 5' cap structure of RNA substrates, and this interaction is greatly stimulated by a poly(A) tail. Several observations suggest that cap-DAN interactions are functionally important for the networking between regulated mRNA stability and translation. First, uncapped RNA substrates are inefficiently deadenylated. Second, a stem-loop structure in the 5' UTR dramatically reduces deadenylation by interfering with cap-DAN interactions. Third, the addition of cap binding protein eIF4E inhibits deadenylation in vitro. These data provide insights into the early steps of substrate recognition that target an mRNA for degradation.

Eukaryotic Initiation Factor-4E↗

PubMed2002

Hypermethylation of the cap structure of both yeast snRNAs and snoRNAs requires a conserved methyltransferase that is localized to the nucleolus.

The m(7)G caps of most spliceosomal snRNAs and certain snoRNAs are converted posttranscriptionally to 2,2,7-trimethylguanosine (m(3)G) cap structures. Here, we show that yeast Tgs1p, an evolutionarily conserved protein carrying a signature of S-AdoMet methyltransferase, is essential for hypermethylation of the m(7)G caps of both snRNAs and snoRNAs. Deletion of the yeast TGS1 gene abolishes the conversion of the m(7)G to m(3)G caps and produces a cold-sensitive splicing defect that correlates with the retention of U1 snRNA in the nucleolus. Consistently, Tgs1p is also localized in the nucleolus. Our results suggest a trafficking pathway in which yeast snRNAs and snoRNAs cycle through the nucleolus to undergo m(7)G cap hypermethylation.

Amino Acid Motifs↗

PubMed2004

Structure and mechanism of mRNA cap (guanine-N7) methyltransferase.

A suite of crystal structures is reported for a cellular mRNA cap (guanine-N7) methyltransferase in complex with AdoMet, AdoHcy, and the cap guanylate. Superposition of ligand complexes suggests an in-line mechanism of methyl transfer, albeit without direct contacts between the enzyme and either the N7 atom of guanine (the attacking nucleophile), the methyl carbon of AdoMet, or the sulfur of AdoMet/AdoHcy (the leaving group). The structures indicate that catalysis of cap N7 methylation is accomplished by optimizing proximity and orientation of the substrates, assisted by a favorable electrostatic environment. The enzyme-ligand structures, together with new mutational data, fully account for the biochemical specificity of the cap guanine-N7 methylation reaction, an essential and defining step of eukaryotic mRNA synthesis.

Alanine↗

PubMed1983

Translational recognition of messenger ribonucleic acid caps as a function of pH.

The degree to which cell-free translation of eukaryotic mRNA is stimulated by the presence of a 5'-terminal 7-methylguanosine-containing cap is affected by a variety of factors including ionic strength, temperature, mRNA concentration, and the type of mRNA. In this report, we show that pH also affects cap dependence. Translation of globin mRNA from which the cap had been enzymatically removed was relatively insensitive to pH compared with capped mRNA. Also, at low pH (6.6-7.2), the cap analogue m7GTP caused little inhibition of globin mRNA translation in a cell-free system whereas at higher pH the degree of inhibition increased. Finally, the overall extent to which globin mRNA translation could be inhibited at saturating concentrations of m7GTP increased with increasing pH. It is also shown that the pKa of the N-1 proton of m7GTP is affected by mono- and divalent cations. At the K+ and Mg2+ concentrations optimal for cell-free translation, the pKa is approximately 7.4. Since the pH optimum for translation is near 7.6, both keto and enolate forms of m7G are present in appreciable amounts. One interpretation for the observed change in cap dependence with pH is that only the enolate form of m7G is recognized by the cap-binding protein.

Animals↗

PubMed1984

Synthesis and biological activity of 5'-capped derivatives of 5'-triphosphoadenylyl(2'----5')adenylyl(2'----5')adenosine.

The oligonucleotides A5'pp5'A2'p5'A2'p5'A and A5'ppp5'A2'p5'A2'p5'A were prepared by reaction of AMP or ADP, respectively, with the 5'-(phosphoimidazolidate) of A2'p5'A2'p5'A. A5'pppp5'A2'(p5'A)n (n = 1-3) were synthesized by reaction of p5'A2'(p5'A)n (n = 1-3) with adenosine 5'-trimetaphosphate. All structures were confirmed by enzyme digestion and 1H and 31P nuclear magnetic resonance (NMR). The products A5'pppp5'A2'p5'A and A5'pppp5'A2'p5'A2'p5'A were found to be identical with two of the products of the 2-5A synthetase catalyzed reaction of Ap4A with ATP, thus confirming the structural assignments made by earlier investigators. In extracts of mouse L cells programmed with encephalomyocarditis virus RNA, A5'pppp5'A2'p5'A2'p5'A2'p5'A and A5'pppp5'A2'p5'A2'p5'A were equipotent with 2-5A itself as inhibitors of translation. The oligomers A5'ppp5'A2'p5'A2'p5'A and A2'pppp5'A2'p5'A were about 100 times less active than 2-5A, and A5'pp5'A2'p5'A2'p5'A was without translational inhibitory activity. When affinity for the 2-5A-dependent endonuclease was determined (by displacement of 2-5A[32P]pCp from endonuclease), all of the analogues, as well as 2-5A itself, had similar affinities for the endonuclease except for A5'pppp5'A2'p5'A, which was bound approximately 100 times less effectively. Under conditions of the radiobinding assay, A5'pppp5'A2'p5'A2'p5'A was degraded (t1/2 = 2 h) to ATP, ADP, AMP, ppp5'A2'p5'A2'p5'A, and p5'A2'p5'A2'p5'A.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗

PubMed1987

Inhibition of eukaryotic translation by analogues of messenger RNA 5'-cap: chemical and biological consequences of 5'-phosphate modifications of 7-methylguanosine 5'-monophosphate.

New analogues of 7-methylguanosine 5'-monophosphate (m7GMP) were synthesized with modified 5'-phosphate moieties by replacement of -O with -H, -CH3, or -NH2. Additional analogues were synthesized with 8-methyl- or 8-aminoguanine base substitutions or ring-opened ribose (2',3'-diol). These compounds were analyzed by 1H and 31P NMR for solution conformation. In addition, they were also analyzed for biological activity as analogues of mRNA 5'-caps by competition as inhibitors of translation in reticulocyte lysate. Substitution of oxygen on the 5'-monophosphate moiety by -H and -CH3 diminished the activity of the cap analogue as a competitive inhibitor; however, replacement by -NH2 did not diminish the activity of the analogue as an inhibitor. It was inferred from this result that cap binding proteins require a hydrogen bond acceptor as opposed to having an exclusive requirement for a second anionic group on the alpha-phosphate moiety. Inhibition results obtained with C8-substituted m7GMP analogues indicated that the 8-amino derivative was a better inhibitor than the 8-methyl derivative of m7GMP. The former is primarily anti whereas the latter is primarily syn with respect to glycosidic bond conformation. This result further supports the model that the anti conformation is the preferred form of the cap structure for interaction with cap binding proteins. The 2',3'-diol derivative of m7GMP was inactive as an inhibitor of translation.

Animals↗

PubMed1989

A spectroscopic study of the binding of m7GTP and m7GpppG to human protein synthesis initiation factor 4E.

The binding of analogues of the 7-methylguanosine-containing cap, m7GTP and m7GpppG, to eIF-4E from human erythrocytes as a function of pH, temperature, and ionic strength is described. From the pH-dependent binding of m7GTP and m7GpppG to eIF-4E, a new model describing the nature of the cap.eIF-4E interaction is proposed. The thermodynamic values and ionic strength dependence of binding are consistent with a binding site which is primarily hydrophobic. Fluorescence and circular dichroism data indicate that tryptophan residues may be involved in base-stacking interactions with the cap in a somewhat buried environment. The model presented here confirms the earlier proposal [Rhoads et al. (1983) Biochemistry 22, 6084-6088] that the enolate tautomer of the cap is preferred for interaction and further proposes that the interaction is with a protonated amino acid residue, such as histidine, while stacking with an aromatic amino acid, such as tryptophan.

Circular Dichroism↗

PubMed1990

A spectroscopic study of the binding of N-7-substituted cap analogues to human protein synthesis initiation factor 4E.

The binding of N-7-substituted cap analogues to eIF-4E from human erythrocytes is described. Data presented here indicate that there is a correlation between the tightness of binding of these cap analogues to eIF-4E and their potency as inhibitors of protein synthesis. This result indicates that the inhibitory activity of the cap analogues is strictly a function of the affinity of the analogue for eIF-4E under equilibrium conditions. The pH dependence of binding of the cap analogues to eIF-4E indicates that the enolate form of the cap is preferred, as originally postulated by Rhoads et al. [(1983) Biochemistry 22, 6084-6088]. Data indicate that there are differences in the mode of binding of alkyl-substituted and aryl-substituted cap analogues to eIF-4E arising from favorable interactions of the phenyl ring with the guanosine moiety. These differences may explain the enhanced recognition of the aryl-substituted cap analogues by eIF-4E.

Cross-Linking Reagents↗

PubMed1990

Fluorescence study of the binding of m7GpppG and rabbit globin mRNA to protein synthesis initiation factors 4A, 4E, and 4F.

The interactions of protein synthesis initiation factors eIF-4E from human erythrocytes and eIF-4A and eIF-4F from rabbit reticulocytes with the cap analogue m7GpppG and rabbit globin mRNA were investigated. The equilibrium binding constants for the binary complex formation of eIF-4E-eIF-4A, m7GpppG-eIF-4E, m7GpppG-eIF-4F, globin mRNA-eIF-4E, globin mRNA-eIF-4F, and globin mRNA-eIF-4A were measured by direct fluorescence titration experiments. The binding of eIF-4E to globin mRNA was found to be 5.5-fold tighter than its binding to m7GpppG; the binding of eIF-4F for globin mRNA and m7GpppG was similar to that of eIF-4E. Association equilibrium constants were determined for the ternary system mRNA-eIF-4E-eIF-4A; four thermodynamically independent equilibria characterize the system. These equilibrium binding constants were used to calculate coupling free energies, which provided an estimate of the cooperativity of the interaction of eIF-4E, eIF-4A, and mRNA. These coupling energies were all found to be small and positive, indicative of anticooperative binding.

Binding Sites↗

PubMed1990

Phenyl azide substituted and benzophenone-substituted phosphonamides of 7-methylguanosine 5'-triphosphate as photoaffinity probes for protein synthesis initiation factor eIF-4E and a proteolytic fragment containing the cap-binding site.

Three photoactive derivatives of the 7-methylguanosine-containing cap of eukaryotic mRNA were used to investigate protein synthesis initiation factor eIF-4E from human erythrocytes and rabbit reticulocytes. Sensitive and specific labeling of eIF-4E was observed with the previously described probe, [gamma-32P]-gamma-[[(4-benzoylphenyl)methyl]amido]-7-methyl-GTP [Blaas et al. (1982) Virology 116, 339; abbreviated [32P]BPM]. A second probe was synthesized that was an azidophenyltyrosine derivative of m7GTP [( 125I]APTM), the monoanhydride of m7GDP with [125I]-N-(4-azidophenyl)-2-(phosphoramido)-3-(4-hydroxy-3-iodop hen yl) propionamide. This probe allowed rapid and quantitative introduction of radioactivity in the last rather than the first step of synthesis and placed the radioactive label on the protein-proximal side of the weak P-N bond. A dissociation constant of 6.9 microM was determined for [125I]APTM, which is comparable to the published values for m7GTP. m7GTP and APTM were equally effective as competitive inhibitors of eIF-4E labeling with [125I]APTM. Like [32P]BPM, [125I]APTM labeled both the full-length (25 kDa) polypeptide and a 16-kDa degradation product, designated eIF-4E*, with labeling occurring in proportion to the amounts of each polypeptide present. A third probe, an azidophenylglycine derivative of m7GTP [( 32P]APGM), the monoanhydride of m7GDP with [32P]-N-(4-azidophenyl)-2-(phosphoramido)acetamide, was also synthesized and shown to label eIF-4E specifically. Unlike [32P]BPM and [125I]APTM, however, [32P]APGM labeled eIF-4E* approximately 4-fold more readily than intact eIF-4E. Tryptic and CNBr cleavage suggested that eIF-4E* consists of a protease-resistant core of eIF-4E that retains the cap-binding site and consists of approximately residues 47-182.

Affinity Labels↗

PubMed1980

Initiation of reovirus transcription by inosine 5'-triphosphate and properties of 7-methylinosine-capped, inosine-substituted messenger ribonucleic acids.

Inosine 5'-triphosphate (ITP) can be utilized in place of guanosine 5'-triphosphate (GTP) for both the initiation and the elongation steps of reovirus transcription, resulting in the synthesis of mRNAs containing 5'-terminal m7IpppIm and internal pI. The apparent molecular weights of the I-substituted products were altered as a consequence of the absence of G-C base pairs and accompanying loss of ordered structure. The migration of I-substituted RNAs in agarose gels and glycerol gradients was similar to glyoxal-treated transcripts; i.e., it decreased 2-fold as compared to the corresponding untreated G-containing mRNAs. 7-Methylinosine-capped (m7I-capped), I-substituted transcripts readily attached to wheat germ 80S ribosomes. Unlike native G-containing mRNAs, they also formed heavier complexes that sedimented faster than 80S complexes even in the presence of the nonhydrolyzable ATP analogue AMPP(NH)P and elongation inhibitor sparsomycin. I-substituted molecules that were capped posttranscriptionally to form m7G-capped 5' ends yielded mostly 80S monosomes, consistent with a strong influence of 5'-terminal structure on initiation of translation. Under limited conditions of initiation, I-substituted RNAs outcompeted G-containing transcripts for ribosome attachment. Although the results are consistent with enhanced binding and freer movement of ribosomes on unstructured templates, synthesis of acid-precipitable polypeptides in wheat germ extract directed by I-substituted RNAs was 15-fold less than with G-containing mRNAs.

Binding Sites↗