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PubMed1980

Presence of cap structures in the messenger RNA of mouse eggs.

The presence of 5'-terminal 7-methylguanosine cap structures in the messenger RNA molecules in mouse eggs was assessed by (1) translational inhibition in a message-dependent reticulocyte cell-free system with the cap analogue 7-methylguanosine-5'-triphosphate, and (2) enzymatic removal of cap strucures with tobacco acid phosphatase followed by end-labelling with gamma-32P-ATP and polynucleotide kinase. The results suggest that the majority of mRNA molecules contain cap structures. There was no detectable difference in degree of capping in unfertilized versus fertilized egg mRNA.

Acid Phosphatase↗
PubMed1981

Shutoff of neuroblastoma cell protein synthesis by Semliki Forest virus: loss of ability of crude initiation factors to recognize early Semliki Forest virus and host mRNA's.

A crude ribosomal wash containing the initiation factors of protein synthesis was isolated from mouse neuroblastoma cells 8 h after infection with Semliki Forest virus (SFV). The activity of this wash was compared with that of a wash from control cells in a cell-free protein-synthesizing "pH5" system, with early SFV mRNA (42S), late SFV mRNA (26S), encephalomyocarditis virus (EMC) mRNA, or neuroblastoma polyadenylated mRNA templates. A pronounced loss of activity (+/-80%) of the crude ribosomal wash from infected cells was observed with host mRNA (neuroblastoma polyadenylated mRNA) and early SFV mRNA, messengers which contain a cap structure at the 5' terminus. However, these washes were only slightly less active in systems programmed with (noncapped) EMC mRNA and late SFV mRNA. Although late SFV mRNA (26S) is capped, the synthesis of late (= structural) proteins in infected lysates was insensitive to inhibition by cap analogs. Purified initiation factors eIF-4B (M(r), 80,000) and cap-binding protein (M(r), 24,000) from reticulocytes (but none of the others) were able to restore the activity of infected factors to about 90% of control levels in systems programmed with early SFV mRNA and host mRNA. These observations indicate that infection-exposed crude initiation factors have a decreased level of eIF-4B and cap-binding protein activity. However, after partial purification of these and other initiation factors from infected and control cells, we found no significant difference in activity when model assay systems were used. Furthermore, both eIF-4B and cap-binding protein from infected cells were able to restore the activity of these infection-exposed factors to the same level obtained when these factors isolated from control cells or reticulocytes were added. A possible mechanism for the shutoff of host cell protein synthesis is discussed.

Animals↗
PubMed1986

Stacking and hydrogen bonding interactions between phenylalanine and guanine nucleotide: crystal structure of L-phenylalanine-7-methylguanosine-5'-monophosphate complex.

The crystal structure of title complex has been analyzed by X-ray diffraction method as a model for elucidating the possible interaction between the phenylalanyl residue of proteins and the N7-protonated or methylated guanine base of nucleic acids. The guanine base is associated with the benzene ring of phenylalanine by stacking interaction, and further connected with the carboxyl group by the formation of a pair of hydrogen bonds. These two interaction modes are suggested to be responsible for the specific recognition of base sequence by protein.

Carrier Proteins↗
PubMed1988

A possible recognition mode of mRNA cap terminal structure by peptide: cooperative stacking and hydrogen-bond pairing interactions between m7GpppA and Trp-Leu-Glu.

1H-NMR and fluorescence spectroscopic studies on the interaction between the Trp-Leu-Glu and m7GpppA have shown a specific binding mode, in which the pi-pi stacking interaction of the Trp indole ring and the hydrogen-bond pairing of Glu carboxyl side group with 7-methylguanine base are simultaneously formed.

Base Composition↗
PubMed1996

1H-NMR studies on association of mRNA cap-analogues with tryptophan-containing peptides.

1H-NMR spectroscopy was applied to a study of the mode of interaction, in aqueous medium in the pH range 5.2-8.5 and at low and high temperatures, between several mono- and dinucleotide analogues of the mRNA cap m7GpppG and a selected tripeptide Trp-Leu-Glu, and a tetrapeptide Trp-Glu-Asp-Glu, the sequence of which corresponds to one of the suspected binding sites in the mRNA cap-binding protein (CBP). A program, GEOSHIFT, was developed, based on ring-current anisotropy theory, for analysis of experimentally observed changes in chemical shifts accompanying interactions between aromatic heterocyclic rings. This permitted quantitative evaluation of stacking interactions between the m7G cap and the tryptophan indole ring, and the relative orientations of the planes of the two rings, spaced about 3.2 angstroms apart. The structures of the stacked complexes were determined. In particular, stacking between m(2,2,7)3G (which has no free amino group for hydrogen bonding) and the indole ring is weaker and quite different from that between m7G and m(2,7)2G and indole. With the dinucleotide cap-analogues, only the m7G component stacks with the indole ring, without disruption of intramolecular stacking. In contrast to numerous earlier reports, the calculated stacking interactions are quantitatively in accord with the values derived from fluorescence measurements. It also has been shown that the positively charged (cationic) form of m7G stacks much more efficiently with the indole ring than the zwitterionic form resulting from dissociation of the guanine ring N1H (pKa approximately 7.3).

Amino Acid Sequence↗
PubMed2006

Crystal structures of U8 snoRNA decapping nudix hydrolase, X29, and its metal and cap complexes.

X29, a 25 kDa Nudix hydrolase from Xenopus laevis that cleaves 5' caps from U8 snoRNA, crystallizes as a homodimeric apoenzyme. Manganese binds crystals of apo-X29 to form holo-X29 only in the presence of nucleot(s)ide. Structural changes in X29 on nucleo-t(s)ide-assisted Mn(+2) uptake account for the observed cooperativity of metal binding. Structures of X29 with GTP or m7GpppA show a different mode of ligand binding from that of other cap binding proteins and suggest a possible three- or four-metal Nudix reaction mechanism. The X29 dimer has no known RNA binding motif, but its striking surface dipolarity and unique structural features create a plausible RNA binding channel on the positive face of the protein. Because U8 snoRNP is essential for accumulation of mature 5.8S and 28S rRNA in vertebrate ribosome biogenesis, and cap structures are required for U8 stability in vivo, X29 could profoundly influence this fundamental cellular pathway.

Amino Acid Sequence↗
PubMed2002

Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins.

mRNA 5'-cap recognition by the eukaryotic translation initiation factor eIF4E has been exhaustively characterized with the aid of a novel fluorometric, time-synchronized titration method, and X-ray crystallography. The association constant values of recombinant eIF4E for 20 different cap analogues cover six orders of magnitude; with the highest affinity observed for m(7)GTP (approximately 1.1 x 10(8) M(-1)). The affinity of the cap analogues for eIF4E correlates with their ability to inhibit in vitro translation. The association constants yield contributions of non-covalent interactions involving single structural elements of the cap to the free energy of binding, giving a reliable starting point to rational drug design. The free energy of 7-methylguanine stacking and hydrogen bonding (-4.9 kcal/mol) is separate from the energies of phosphate chain interactions (-3.0, -1.9, -0.9 kcal/mol for alpha, beta, gamma phosphates, respectively), supporting two-step mechanism of the binding. The negatively charged phosphate groups of the cap act as a molecular anchor, enabling further formation of the intermolecular contacts within the cap-binding slot. Stabilization of the stacked Trp102/m(7)G/Trp56 configuration is a precondition to form three hydrogen bonds with Glu103 and Trp102. Electrostatically steered eIF4E-cap association is accompanied by additional hydration of the complex by approximately 65 water molecules, and by ionic equilibria shift. Temperature dependence reveals the enthalpy-driven and entropy-opposed character of the m(7)GTP-eIF4E binding, which results from dominant charge-related interactions (DeltaH degrees =-17.8 kcal/mol, DeltaS degrees= -23.6 cal/mol K). For recruitment of synthetic eIF4GI, eIF4GII, and 4E-BP1 peptides to eIF4E, all the association constants were approximately 10(7) M(-1), in decreasing order: eIF4GI>4E-BP1>eIF4GII approximately 4E-BP1(P-Ser65) approximately 4E-BP1(P-Ser65/Thr70). Phosphorylation of 4E-BP1 at Ser65 and Thr70 is insufficient to prevent binding to eIF4E. Enhancement of the eIF4E affinity for cap occurs after binding to eIF4G peptides.

Adaptor Proteins, Signal Transducing↗
PubMed1989

Inhibition of eukaryotic translation by nucleoside 5'-monophosphate analogues of mRNA 5'-cap: changes in N7 substituent affect analogue activity.

Nucleotide cap analogues of 7-methylguanosine 5'-monophosphate (m7GMP) were synthesized in which the 7-methyl moiety was replaced with 7-ethyl (e7), 7-propyl (p7), 7-isopropyl (ip7), 7-butyl (b7), 7-isobutyl (ib7), 7-cyclopentyl (cp7), 7-(carboxymethyl) (cm7), 7-benzyl (bn7), 7-(2-phenylethyl) [7-(2-PhEt)], and 7-(1-phenylethyl) [7-(1-PhEt)]. These derivatives were assayed as competitive inhibitors of capped mRNA translation in reticulocyte lysate. We observed that N7 alkyl and alicyclic substituents larger than ethyl significantly decreased the inhibitory activity of these cap analogues presumably by decreasing their affinity for cap binding proteins, which participate in the initiation of translation. This result defined a maximum size for this class of N7 substituents in the nucleotide binding domain of cap binding proteins. Like m7GMP, the N7-substituted GMP derivatives synthesized in this study were found to be predominantly in the anti conformation as determined by proton NMR analyses. However, bn7GMP and 7-(2-PhEt)GMP, which have aromatic N7 substituents, were more effective than m7GMP as competitive inhibitors of translation. The increased affinity of bn7GMP for cap binding proteins was further examined by synthesis of beta-globin mRNA containing 5'-bn7G, 5'-m7G, or 5'-e7G cap structures. These modified mRNAs were tested as translation templates. Messenger RNA capped with bn7G was observed to increase the translation activity of the template 1.8-fold relative to that of its m7G-capped mRNA counterpart. By contrast, e7G-capped mRNA was 25% less active than m7G-capped mRNA.2+V photo-cross-linking of m7G-capped mRNA to cap binding proteins

Animals↗
PubMed2003

Quantitative analysis of influenza virus RNP interaction with RNA cap structures and comparison to human cap binding protein eIF4E.

Influenza virus polymerase uses capped RNA primers for transcription initiation in infected cells. This unique mechanism involves the specific binding of the polymerase to capped mRNA precursors in the nucleus of infected cells. These host RNAs are then cleaved by a polymerase associated endonuclease at a position 10-15 nucleotides downstream of the cap structure. The resulting capped RNA oligonucleotides function as primers for transcription initiation. The viral cap binding site has previously been mapped to the PB2 subunit of the trimeric influenza polymerase complex. We have established a quantitative assay system for the analysis of cap interaction with PB2 as part of the native, viral ribonucleoprotein complex (RNP) using a specific UV cross-linking approach. Cap binding was not affected by the RNase pretreatment of the capped RNA substrate and cap binding was not inhibited by excess uncapped RNA, indicating that under the assay conditions, the majority of the binding energy was contributed by the interaction with the cap structure. Binding to 7-methyl-GTP was found to involve synergistic interaction with 7-methyl guanosine and triphosphate binding subsites. A similar mode of interaction with 7-methyl-GTP was found for human cap binding protein eIF4E. However, the potency of 7-methyl-GTP for cap binding inhibition was 200-fold stronger with eIF4E and had a higher contribution from the triphosphate moiety as compared to influenza RNP. Due to this difference in cap subsite interaction, it was possible to identify novel cap analogues, which selectively interact with influenza virus, but not human cap binding protein.

Base Sequence↗
PubMed2005

Translation initiation factor (eIF) 4B affects the rates of binding of the mRNA m7G cap analogue to wheat germ eIFiso4F and eIFiso4F.PABP.

Previous kinetic binding studies of wheat germ protein synthesis eukaryotic translational initiation factor eIFiso4F and its subunit, eIFiso4E, with m(7)GTP and mRNA analogues indicated that binding occurred by a two-step process with the first step occurring at a rate close to the diffusion-controlled rate [Sha, M., Wang, Y., Xiang, T., van Heerden, A., Browning, K. S., and Goss, D. J. (1995) J. Biol. Chem. 270, 29904-29909]. The kinetic effects of eIF4B, PABP, and wheat germ eIFiso4F with two mRNA cap analogues and the temperature dependence of this reaction were measured and compared. The Arrhenius activation energies for binding of the two mRNA cap analogues, Ant-m(7)GTP and m(7)GpppG, were significantly different. Fluorescence stopped-flow studies of the eIFiso4F.eIF4B protein complex with two m(7)G cap analogues show a concentration-independent conformational change. The rate of this conformational change was approximately 2.4-fold faster for the eIFiso4F.eIF4B complex compared with our previous studies of eIFiso4F [Sha, M., Wang, Y., Xiang, T., van Heerden, A., Browning, K. S., and Goss, D. J. (1995) J. Biol. Chem. 270, 29904-29909]. The dissociation rates were 3.7- and 5.4-fold slower for eIFiso4F.Ant-m(7)GTP and eIFiso4F.m(7)GpppG, respectively, in the presence of eIF4B and PABP. These studies show that eIF4B and PABP enhance the interaction with the cap and probably are involved in protein-protein interactions as well. The temperature dependence of the cap binding reaction was markedly reduced in the presence of either eIF4B or PABP. However, when both eIF4B and PABP were present, not only was the energy barrier reduced but the binding rate was faster. Since cap binding is thought to be the rate-limiting step in protein synthesis, these two proteins may perform a critical function in regulation of the overall protein synthesis efficiency. This suggests that the presence of both proteins leads to a rapid, stable complex, which serves as a scaffold for further initiation complex formation.

Dinucleoside Phosphates↗
PubMed1990

A nuclear cap binding protein from HeLa cells.

We have identified a cap binding protein in a HeLa nuclear extract using a gel mobility shift assay probed with capped RNA. Subcellular fractionation of HeLa cells revealed that the majority (about 70%) of the cap binding activity is present in the nuclear extract, about 20% is in the cytoplasmic S100 fraction, and almost none in the ribosome-high salt wash fraction, indicating that the protein in active form localizes mainly in the nuclei. Competition experiments with various cap analogues showed that the G(5')ppp(5')N-blocking structure as well as the methyl residue at the N7 position of the blocking guanosine is important for the binding of this protein, and that the trimethylguanosine cap structure which exists at the 5' termini of many snRNAs is not recognized by this protein. Immunoprecipitation experiments using various anti-snRNP antibodies suggested that this protein is partially associated with U2 snRNP. We purified this protein to near homogeneity from a HeLa nuclear extract by several chromatographic procedures including capped RNA-Sepharose chromatography. The purified protein shows molecular weight of 80 kilodaltons, as judged by SDS gel electrophoresis, and binds specifically to the cap structure.

Binding, Competitive↗
PubMed2005

Synthesis and biochemical properties of novel mRNA 5' cap analogs resistant to enzymatic hydrolysis.

A series of new dinucleotide cap analogs with methylene groups replacing oxygens within the pyrophosphate moieties have been synthesized. All the compounds were resistant to the human scavenger decapping hydrolase, DcpS. Binding constants of the modified caps to eIF4E are comparable to those obtained for m7GpppG. This suggests these methylene modifications in the pyrophosphate chain do not significantly affect cap-binding at least for eIF4E. These cap analogs are also good inhibitors of in vitro translation. mRNAs capped with novel analogs were translated similarly to the mRNA capped with the parent m7GpppG.

Animals↗
PubMed1996

Dinucleoside 5', 5"'-P1, P3-triphosphate hydrolase from yellow lupin (Lupinus luteus) seeds: purification to homogeneity and hydrolysis of mRNA 5'-cap analogs.

Three separable forms of diadenosine 5',5"'-P1, P3-triphosphate (Ap3A)-degrading activity were revealed when proteins obtained from the meal of yellow lupin seeds by ammonium sulfate precipitation were chromatographed on a DEAE-Sephacel column. The major form, which eluted first at the lowest salt concentration (0.15 M KCl), was free of any activity converting the reaction products, ADP and AMP. Its further purification by gel filtration on Sephadex G-200 and by affinity elution from an AMP-agarose column yielded homogeneous protein as demonstrated on SDS-polyacrylamide gel electrophorograms. The enzyme is a single polypeptide chain of M(r) 41 kDa. Eleven guanosine-containing dinucleoside triphosphates, including analogs of the mRNA 5'-cap structure, have been tested as potential substrates of the lupin "Ap3A hydrolase." All have been hydrolyzed yielding mixtures of corresponding nucleoside mono- and diphosphates. Asymmetrical compounds gave four products; m7Gp3G, et7Gp3G, and bz7Gp3G were hydrolyzed randomly, whereas m7Gp3A, m7Gp3C, and m7Gp3U yielded at least 80% m7GMP plus corresponding NDP and 20% or less NMP plus m7GDP. Hydrolysis of the guanosine-containing hybrids, Ap3G, Cp3G, and Up3G, yielded at least 85% GMP plus corresponding NDP. All dinucleotides containing the m7G-moiety were hydrolyzed 2- to 4.5-fold faster than Ap3A. Thus a general name, "dinucleoside triphosphate hydrolase," is more appropriate for the enzymatic activity described.

Acid Anhydride Hydrolases↗
PubMed1985

1,7-Diethylguanosine formation in tRNA chemical ethylation by ethionine and ethylnitrosourea.

The mechanism of ethionine carcinogenesis and more generally the relationship between alkylation of nucleic acids by chemical carcinogens and oncogenesis still remain obscure. In the present study the rat liver tRNA ethylation by L-[ethyl-1-3H]ethionine was reinvestigated by examining in particular the highly radioactive 'pyrimidine-nucleotide-like' fraction found earlier in acid hydrolysates of hepatic tRNA from ethionine-treated rats. The following results were obtained: (1) ultraviolet-spectral and chromatographic analyses showed the presence of 1,7-diethylguanosine in this 'pyrimidine-nucleotide-like' fraction; (2) the dialkyl compound was recovered exclusively in the form of imidazole-ring-opened derivatives. When [1-14C]ethylnitrosourea was used as alkylating agent, the in vivo ethylation pattern of tRNA from various organs of rat showed an analogous radioactive 'pyrimidine-nucleotide-like' fraction as main radioactive product. On the contrary, tRNA ethylation pattern after in vitro reaction with [1-14C]ethylnitrosourea exhibited a main radioactivity peak (85% of the total radioactivity recovered) in coincidence of the chromatographic area of 1,7-diethylguanine. The 1,7-diethylguanosine moieties of tRNA were extremely labile both under physiological and alkaline conditions. The 1,7-diethylguanine-associated radioactivity was completely lost from [14C]ethyl-tRNA after only 7 h incubation at 37 degrees C and pH 7.3, while at pH 11.4 this process was preceded by the conversion of the 1,7-diethylguanosine residues into imidazole-ring-opened derivatives.

Alkylating Agents↗
PubMed2000

Kinetic proofreading scanning models for eukaryotic translational initiation: the cap and poly(A) tail dependency of translation.

Two simplified kinetic proofreading scanning (KPS) models were proposed to describe the 5' cap and 3' poly(A) tail dependency of eukaryotic translation initiation. In Model I, the initiation factor complex starts scanning and unwinding the secondary structure of the 5' untranslated region (UTR) from the 5' terminus of mRNA. In Model II, the initiation factor complex starts scanning from any binding site in the 5' UTR. In both models, following ATP hydrolysis, the initiation factor complex either dissociates from mRNA or continues to scan and unwind RNA secondary structure in the 5' UTR. This step repeats n times until the AUG codon is reached. These two models show very different cap and/or poly(A) tail dependency of translation initiation. The models predict that both cap and poly(A) tail dependencies of translation, and translatability of mRNAs are coupled with the structure of 5' UTR: the translation of mRNA with structured 5' UTR is strongly cap- and poly(A) tail-dependent; while translation of mRNA with unstructured 5' UTR is less cap- and poly(A) tail-dependent. We use these two models to explain: (1) the cap and poly(A) tail dependence of translation; (2) the effect of exogenous poly(A) on translation; (3) repression of host mRNA and translation of late adenovirus mRNA in the late phase of adenovirus infection; (4) repression of host mRNA and translation of Vaccinia virus mRNA in virus-infected cell; (5) heat shock repression of translation of normal mRNA and stimulation of translation of hsp mRNA; and (6) the synergistic effect of cap and poly(A) tail on stimulating translation. The kinetic proofreading scanning models provide a coherent interpretation of those phenomena.

5' Untranslated Regions↗