guanosine-diphosphate and 7-methylguanosine-5--diphosphate

The eukaryotic cap and poly(A) tail binding proteins, eIF4E and Pab1p, play important roles in the initiation of protein synthesis. The recent structures of the complex of eIF4E bound to the methylated guanosine (cap) found at the 5'end of messenger RNA (mRNA), the complex of eIF4E bound to peptide fragments of two related translation factors (eIF4G and 4E-BP1), and the complex of the N-terminal fragment of Pab1p bound to polyadenylate RNA have revealed that eIF4E and Pab1p contain at least two distinct functional surfaces. One surface is used for binding mRNA, and the other for binding proteins involved in translation initiation.

Topics: Amino Acid Sequence; Animals; Eukaryotic Cells; Eukaryotic Initiation Factor-4E; Guanosine Diphosphate; Models, Molecular; Peptide Initiation Factors; Poly A; Poly(A)-Binding Proteins; Protein Binding; Protein Biosynthesis; Protein Conformation; RNA-Binding Proteins

This article describes a simple, reliable, efficient, and general method for the synthesis of 7-methylguanosine nucleotides such as 7-methylguanosine 5'-O-monophosphate (m

Topics: Guanosine Diphosphate; Indicators and Reagents; Methylation; RNA Cap Analogs; Solvents; Sulfuric Acid Esters

Elimination of the 5' cap of eukaryotic mRNAs, known as decapping, is considered to be a crucial, irreversible and highly regulated step required for the rapid degradation of mRNA by Xrn1, the major cytoplasmic 5'-3' exonuclease. Decapping is accomplished by the recruitment of a protein complex formed by the Dcp2 catalytic subunit and its Dcp1 cofactor. However, this complex has a low intrinsic enzymatic activity and requires several accessory proteins such as the Lsm1-7 complex, Pat1, Edc1-Edc2 and/or Edc3 to be fully active. Here we present the crystal structure of the active form of the yeast Kluyveromyces lactis Dcp1-Dcp2 enzyme bound to its product (m

Topics: Crystallography, X-Ray; Endoribonucleases; Enzyme Activation; Fungal Proteins; Guanosine Diphosphate; Models, Molecular; Protein Binding; Protein Conformation; RNA Stability; Saccharomycetales

Eukaryotic 5' mRNA cap structures participate to the post-transcriptional control of gene expression before being released by the two main mRNA decay pathways. In the 3'-5' pathway, the exosome generates free cap dinucleotides (m7GpppN) or capped oligoribonucleotides that are hydrolyzed by the Scavenger Decapping Enzyme (DcpS) forming m7GMP. In the 5'-3' pathway, the decapping enzyme Dcp2 generates m7GDP. We investigated the fate of m7GDP and m7GpppN produced by RNA decay in extracts and cells. This defined a pathway involving DcpS, NTPs and the nucleoside diphosphate kinase for m7GDP elimination. Interestingly, we identified and characterized in vitro and in vivo a new scavenger decapping enzyme involved in m7GpppN degradation. We show that activities mediating cap elimination identified in yeast are essentially conserved in human. Their alteration may contribute to pathologies, possibly through the interference of cap (di)nucleotide with cellular function.

Topics: Acid Anhydride Hydrolases; Adenosine Triphosphate; Dinucleoside Phosphates; Endoribonucleases; Guanosine Diphosphate; HEK293 Cells; Humans; N-Glycosyl Hydrolases; Neoplasm Proteins; Nucleoside-Diphosphate Kinase; RNA Cap Analogs; RNA Caps; RNA Stability; RNA, Messenger; Saccharomyces cerevisiae Proteins

The eukaryotic initiation factor 4E (eIF4E) is the key component of the translational initiation complex that recruits mRNA by binding to a unique "cap" structure located at the 5' end of the mRNA. Overexpression of eIF4E has been implicated in the development of cancer, potentially as a result of increasing the cellular levels of proteins involved in processes that include proliferation and regulation of apoptosis. As a result, the cap-binding site of eIF4E has become a target for the development of anti-cancer therapeutics. The structure of eIF4E bound to the cap mimic 7-methyl-GDP revealed that two tryptophans from different loops in eIF4E sandwiched the 7-methylguanine group between them. This interaction gives rise to a strong exciton coupling signal between the two tryptophans that can be visualized by CD spectroscopy. eIF4E is a challenging protein to work with because of a propensity to aggregate under conditions used in biophysical techniques. CD spectroscopy provides a gentle, solution-based approach to study binding to the cap-binding site of eIF4E. Evidence is provided that the exciton coupling signal can be used to both qualitatively and quantitatively analyze the binding of cap analogs to eIF4E.

Topics: Binding Sites; Circular Dichroism; Eukaryotic Initiation Factor-4E; Guanosine Diphosphate; Nucleotides; Protein Binding; Protein Refolding; Protein Structure, Tertiary; Recombinant Proteins; Ribavirin; RNA Cap Analogs; RNA Caps; Solutions

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' → 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' → 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

Topics: Amino Acid Sequence; Animals; Caenorhabditis elegans; Endoribonucleases; Escherichia coli; Gene Expression Regulation, Enzymologic; Guanine Nucleotides; Guanosine Diphosphate; Humans; Hydrolysis; Molecular Sequence Data; RNA, Messenger; Saccharomyces cerevisiae; Species Specificity

The Arabidopsis thaliana decapping enzyme (AtDcp2) was characterized by bioinformatics analysis and by biochemical studies of the enzyme and mutants produced by recombinant expression. Three functionally significant regions were detected: (i) a highly disordered C-terminal region with a putative PSD-95, Discs-large, ZO-1 (PDZ) domain-binding motif, (ii) a conserved Nudix box constituting the putative active site and (iii) a putative RNA binding domain consisting of the conserved Box B and a preceding loop region. Mutation of the putative PDZ domain-binding motif improved the stability of recombinant AtDcp2 and secondary mutants expressed in Escherichia coli. Such recombinant AtDcp2 specifically hydrolysed capped mRNA to produce 7-methyl GDP and decapped RNA. AtDcp2 activity was Mn(2+)- or Mg(2+)-dependent and was inhibited by the product 7-methyl GDP. Mutation of the conserved glutamate-154 and glutamate-158 in the Nudix box reduced AtDcp2 activity up to 400-fold and showed that AtDcp2 employs the catalytic mechanism conserved amongst Nudix hydrolases. Unlike many Nudix hydrolases, AtDcp2 is refractory to inhibition by fluoride ions. Decapping was dependent on binding to the mRNA moiety rather than to the 7-methyl diguanosine triphosphate cap of the substrate. Mutational analysis of the putative RNA-binding domain confirmed the functional significance of an 11-residue loop region and the conserved Box B.

Topics: Amino Acid Motifs; Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins; Computational Biology; Endoribonucleases; Escherichia coli; Fluorides; Glutamic Acid; Guanosine Diphosphate; Histidine; Lysine; Molecular Sequence Data; Mutation; Phosphines; Protein Structure, Tertiary; Recombinant Fusion Proteins; RNA, Messenger; Sequence Deletion

Vaccinia virus (VACV) encodes enzymes that cap the 5' end of viral mRNAs, which enhances their stability and translation. Nevertheless, recent studies demonstrated that the VACV D10 protein (VACV-WR_115) decaps mRNA, an enzymatic activity not previously shown to be encoded by a virus. The decapping activity of D10 is dependent on a Nudix hydrolase motif that is also present in the VACV D9 protein (VACV-WR_114), which shares 25% sequence identity with D10. Here, we showed that a purified recombinant VACV D9 fusion protein also decaps mRNA and that this activity was abolished by point mutations in the Nudix hydrolase motif. Decapping was specific for a methylated cap attached to RNA and resulted in the liberation of m7GDP. D9 differed from D10 in requiring a longer capped RNA substrate for optimal activity, having greater sensitivity to inhibition by uncapped RNA, and having lower sensitivity to inhibition by nucleotide cap analogs unattached to RNA. Since D9 is expressed early in infection and D10 late, we suggest that the two proteins enhance mRNA turnover and manipulate gene expression in a complementary and overlapping manner.

Topics: Amino Acid Motifs; Endoribonucleases; Enzyme Inhibitors; Guanosine Diphosphate; Mutagenesis, Site-Directed; Mutation, Missense; Point Mutation; Recombinant Fusion Proteins; RNA Caps; Substrate Specificity; Vaccinia virus; Viral Proteins

We report an industrial scale facile synthesis of 7-methyl guanosine 5'-diphosphate, which plays an important role in synthesis of various mRNA cap analogs. An efficient and selective methylation at position 7 of guanosine 5'-diphosphate was achieved by dissolving guanosine 5'-diphosphate in water and drops wise addition of dimethyl sulfate over a period of 1 h at room temperature. The reaction was completed within 2 h and resulted in more than a 96% yield. The desired product, 7-methyl GDP was purified by using BPG column on AKTA Purifier 100. Certainly, this method has advantages over the known methylation method, in terms of yield, economy, safety, and environmental concerns.

Topics: Guanosine Diphosphate; Methylation; RNA Cap Analogs; RNA, Messenger

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.

Topics: Apoproteins; Crystallography, X-Ray; Dimerization; Endoribonucleases; Guanosine Diphosphate; Humans; Ligands; Models, Molecular; Mutation; Pliability; Protein Structure, Tertiary; RNA Cap Analogs; RNA Caps; RNA, Messenger; Solvents; Static Electricity; Structure-Activity Relationship; Substrate Specificity; Tyrosine

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.

Topics: Animals; Binding Sites; Eukaryotic Initiation Factor-4E; Fluorescence; Guanosine Diphosphate; Guanosine Triphosphate; Organophosphorus Compounds; RNA Cap Analogs; RNA Caps; RNA, Messenger; RNA, Protozoan; RNA, Spliced Leader; Trypanosomatina

Topics: Amino Acid Sequence; Amino Acids; Eukaryotic Initiation Factor-4E; Eukaryotic Initiation Factor-4G; Guanosine Diphosphate; Humans; Nuclear Magnetic Resonance, Biomolecular

Eukaryotic mRNA degradation proceeds through two main pathways, both involving mRNA cap breakdown. In the 3'-5' mRNA decay pathway, mRNA body degradation generates free m7GpppN that is hydrolyzed by DcpS generating m7GMP. In the 5'-3' pathway, the recently identified human Dcp2 decapping enzyme cleaves the cap of deadenylated mRNAs to produce m7GDP and 5'-phosphorylated mRNA. We investigated mRNA decay in human cell extracts by using a new assay for decapping. We observed that 5'-phosphorylated intermediates resulting from decapping appear after incubation of a substrate RNA in human cell extracts, indicating the presence of an active 5'-3' mRNA decay pathway. Surprisingly, however, the cognate m7GDP product was not detected, whereas abundant amounts of m7GMP were generated. Additional experiments revealed that m7GDP is, unexpectedly, efficiently converted to m7GMP in extracts from various organisms. The factor necessary and sufficient for this reaction was identified as DcpS in both yeast and human. m7GMP is thus a general, pathway-independent, by-product of eukaryotic mRNA decay. m7GDP breakdown should prevent misincorporation of methylated nucleotides in nucleic acids and could generate a unique indicator allowing the cell to monitor mRNA decay.

Topics: Animals; Base Sequence; Cell Line; Endoribonucleases; Guanosine Diphosphate; Humans; In Vitro Techniques; Models, Biological; Recombinant Proteins; RNA Caps; RNA Stability; RNA, Messenger; Saccharomyces cerevisiae; Xenopus

A method for extracting kinetic and optical parameters from progress curves for protein-ligand association, obtained by stopped-flow experiments, is described. The method is limited to one-step and two-step association kinetics, but it allows concentration of protein and offset of the signals to be adjustable parameters during an interactive non-linear least-squares fitting procedure. The method is tested on simulated pseudo-experimental data and applied to progress curves obtained in a stopped-flow spectrofluorimeter, for association of the translation initiation factor eIF4E with 7-methyl-GDP, an analog of 5'-end of mRNA.

Topics: Algorithms; Biochemistry; Dose-Response Relationship, Drug; Eukaryotic Initiation Factor-4E; Guanosine Diphosphate; Kinetics; Ligands; Models, Statistical; Peptide Initiation Factors; Protein Binding; RNA, Messenger; Spectrometry, Fluorescence

While decapping plays a major role in mRNA turnover in yeast, biochemical evidence for a similar activity in mammalian cells has been elusive. We have now identified a decapping activity in HeLa cytoplasmic extracts that releases (7me)GDP from capped transcripts. Decapping is activated in extracts by the addition of (7me)GpppG, which specifically sequesters cap-binding proteins such as eIF4E and the deadenylase DAN/PARN. Similar to in vivo observations, the presence of a poly(A) tail represses decapping of RNAs in vitro in a poly(A)-binding protein-dependent fashion. AU-rich elements (AREs), which act as regulators of mRNA stability in vivo, are potent stimulators of decapping in vitro. The stimulation of decapping by AREs requires sequence-specific ARE-binding proteins. These data suggest that cap recognition and decapping play key roles in mediating mRNA turnover in mammalian cells.

Topics: AT Rich Sequence; Binding, Competitive; Cell Extracts; Cytoplasm; Dinucleoside Phosphates; Eukaryotic Initiation Factor-4E; Guanosine Diphosphate; HeLa Cells; Humans; Models, Genetic; Peptide Initiation Factors; Poly A; Poly(A)-Binding Proteins; Regulatory Sequences, Nucleic Acid; RNA Caps; RNA Stability; RNA-Binding Proteins; RNA, Messenger; Saccharomyces cerevisiae

eIF4G uses a conserved Tyr-X-X-X-X-Leu-phi segment (where X is variable and phi is hydrophobic) to recognize eIF4E during cap-dependent translation initiation in eukaryotes. High-resolution X-ray crystallography and complementary biophysical methods have revealed that this eIF4E recognition motif undergoes a disorder-to-order transition, adopting an L-shaped, extended chain/alpha-helical conformation when it interacts with a phylogenetically invariant portion of the convex surface of eIF4E. Inhibitors of translation initiation known as eIF4E-binding proteins (4E-BPs) contain similar eIF4E recognition motifs. These molecules are molecular mimics of eIF4G, which act by occupying the same binding site on the convex dorsum of eIF4E and blocking assembly of the translation machinery. The implications of our results for translation initiation are discussed in detail, and a molecular mechanism for relief of translation inhibition following phosphorylation of the 4E-BPs is proposed.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Animals; Binding Sites; Carrier Proteins; Cell Cycle Proteins; Conserved Sequence; Crystallization; Crystallography, X-Ray; Eukaryotic Initiation Factor-4E; Eukaryotic Initiation Factor-4F; Eukaryotic Initiation Factor-4G; Eukaryotic Initiation Factors; Guanosine Diphosphate; Humans; Mice; Models, Molecular; Molecular Mimicry; Molecular Sequence Data; Peptide Fragments; Peptide Initiation Factors; Phosphoproteins; Phosphorylation; Protein Biosynthesis; Protein Structure, Secondary; RNA Caps; Sequence Deletion

The mRNA cap-binding protein eIF4E is the limiting factor in the eIF4F translation initiation complex, which mediates the binding of the 40S ribosome to the mRNA. 15N relaxation studies have been used to characterize the backbone dynamics of deuterated eIF4E in a CHAPS micelle for the apoprotein, the m7GDP-bound form, and the dinucleotide (m7GpppA)-bound form, as well as for CHAPS-free eIF4E. Large differences in overall correlation time between the CHAPS-free form (11.8 ns) and samples containing different concentrations of CHAPS (15.9-19.4 ns) indicate that eIF4E is embedded in a large micelle in the presence of CHAPS, with a total molecular weight in the range of 40-60 kDa. CHAPS seems to restrict the mobility of the a2-b3 and a4-b5 loops which are thought to be embedded in the micelle. No significant changes in overall mobility were seen between the m7 GDP-bound form, the m7GpppA-bound form, and the apoprotein. Amide hydrogen exchange data indicate the presence of slowly exchanging amides in two surface-exposed helices (a2 and a4), as well as the a4-b5 loop, indicating protection by the CHAPS micelle. The micelle covers the convex side of the protein away from the cap-binding site.

Topics: Cholic Acids; Cloning, Molecular; Detergents; Eukaryotic Initiation Factor-4E; Guanosine Diphosphate; Hydrogen Bonding; Kinetics; Micelles; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Nucleic Acid Conformation; Peptide Initiation Factors; Protein Structure, Secondary; Recombinant Proteins; RNA Cap Analogs; RNA Caps; Saccharomyces cerevisiae

The X-ray structure of the eukaryotic translation initiation factor 4E (eIF4E), bound to 7-methyl-GDP, has been determined at 2.2 A resolution. eIF4E recognizes 5' 7-methyl-G(5')ppp(5')N mRNA caps during the rate-limiting initiation step of translation. The protein resembles a cupped hand and consists of a curved, 8-stranded antiparallel beta sheet, backed by three long alpha helices. 7-methyl-GDP binds in a narrow cap-binding slot on the molecule's concave surface, where 7-methyl-guanine recognition is mediated by base sandwiching between two conserved tryptophans, plus formation of three hydrogen bonds and a van der Waals contact between its N7-methyl group and a third conserved tryptophan. The convex dorsal surface of the molecule displays a phylogenetically conserved hydrophobic/acidic portion, which may interact with other translation initiation factors and regulatory proteins.

Topics: Binding Sites; Conserved Sequence; Crystallography; Escherichia coli; Eukaryotic Cells; Eukaryotic Initiation Factor-4E; Guanosine Diphosphate; Methylation; Molecular Sequence Data; Nucleic Acid Conformation; Peptide Initiation Factors; Protein Biosynthesis; Protein Structure, Secondary; Protein Structure, Tertiary; RNA Cap Analogs; RNA, Messenger; Sequence Homology, Amino Acid

eIF4E, the mRNA cap binding protein, is a master switch that controls eukaryotic translation. To be active, it must bind eIF4G and form the eIF4F complex, which also contains eIF4A. Translation is downregulated by association of eIF4E with 4E-BP, which occupies the eIF4G binding site. Signalling events acting on 4E-BP cause it to dissociate from eIF4E, and eIF4E is then free to bind eIF4G to form the active eIF4F complex. We have solved the structure of the yeast eIF4E/m7Gpp complex in a CHAPS micelle. We determined the position of the second nucleotide in a complex with m7GpppA, and identified the 4E-BP binding site. eIF4E has a curved eight-stranded antiparallel beta-sheet, decorated with three helices on the convex face and three smaller helices inserted in connecting loops. The m7G of the cap is intercalated into a stack of tryptophans in the concave face. The 4E-BP binding site is located in a region encompassing one edge of the beta-sheet, the adjacent helix a2 and several regions of non-regular secondary structure. It is adjacent to, but does not overlap the cap-binding site.

Topics: Amino Acid Sequence; Binding Sites; Carrier Proteins; Cholic Acids; Detergents; Eukaryotic Initiation Factor-4E; Eukaryotic Initiation Factors; Guanosine Diphosphate; Magnetic Resonance Spectroscopy; Micelles; Molecular Sequence Data; Peptide Initiation Factors; Protein Conformation; RNA Cap Analogs; Tryptophan; Yeasts

The X-ray structure of the eukaryotic translation initiation factor 4E (eIF4E), bound to 7-methyl-GDP, has been determined at 2.2A resolution. eIF4E recognizes 5' 7-methyl-G(5')ppp(5')N mRNA caps during the rate-limiting initiation step of translation. The protein resembles a cupped hand, and consists of a curved, 8-stranded antiparallel beta-sheet, backed by three long alpha-helices. 7-methyl-GDP binds in a narrow cap-binding slot on the molecule's concave surface, where 7-methyl-guanine recognition is mediated by base sandwiching between two conserved tryptophans, plus formation of three hydrogen bonds and a van der Waals contact between its N7-methyl group and a third conserved tryptophan. Additional protein-ligand interactions include salt bridges and hydrogen bonds, plus water-mediated hydrogen bonds. The observed mode of 5' m-RNA cap recognition is almost certainly conserved among all known eIF4Es.

Topics: Crystallography, X-Ray; Dinucleoside Phosphates; Eukaryotic Initiation Factor-4E; Guanosine Diphosphate; Hydrogen Bonding; Hydrogen-Ion Concentration; Models, Molecular; Peptide Initiation Factors; Protein Conformation; Protein Structure, Secondary; RNA Caps