ABSTRACT
Recent advances have made it possible to synthesize mRNA in vitro that is relatively stable when introduced into mammalian cells, has a diminished ability to activate the innate immune response against exogenous (virus-like) RNA, and can be efficiently translated into protein. Synthetic methods have also been developed to produce mRNA with unique investigational properties such as photo-cross-linking, fluorescence emission, and attachment of ligands through click chemistry. Synthetic mRNA has been proven effective in numerous applications beneficial for human health such as immunizing patients against cancer and infections diseases, alleviating diseases by restoring deficient proteins, converting somatic cells to pluripotent stem cells to use in regenerative medicine therapies, and engineering the genome by making specific alterations in DNA. This introductory chapter provides background information relevant to the following 20 chapters of this volume that present protocols for these applications of synthetic mRNA.
Subject(s)
Genetic Engineering/methods , RNA, Messenger/genetics , Animals , Gene Transfer Techniques , Humans , Protein Biosynthesis , RNA Stability , RNA, Messenger/chemistry , Synthetic BiologyABSTRACT
Since DNA and histone levels must be closely balanced for cell survival, histone expressions are highly regulated. The regulation of replication-dependent histone expression is mainly achieved at the mRNA level, as the mRNAs are rapidly removed when DNA replication is inhibited during S-phase. Histone mRNA degradation initiates with addition of multiple uridines (oligouridylation) following the 3' stem-loop (SL) catalyzed by terminal uridyltransferase (TUTase). Previous studies showed that histone mRNA degradation occurs through both 5' â 3' and 3' â 5' processes, but the relative contributions are difficult to dissect due to lack of established protocols. The translational efficiency and stability of synthetic mRNA in both cultured cells and whole animals can be improved by structural modifications at the both 5' and 3' termini. In this chapter, we present methods of utilizing modified cap dinucleotide analogs to block 5' â 3' degradation of a reporter mRNA containing canonical histone mRNA 3' SL and monitoring how oligouridylation and 3' â 5' degradation occur. Protocols are presented for synthesis of reporter mRNA containing the histone 3' SL and modified cap analogs, monitoring mRNA stability and unidirectional degradation either from 5' or 3' termini, and detection of oligo(U) tracts from degradation products by either traditional or deep sequencing.
Subject(s)
Histones/genetics , RNA Cap Analogs/metabolism , RNA, Messenger/chemistry , RNA, Messenger/metabolism , HeLa Cells , Humans , Oligoribonucleotides/metabolism , Protein Biosynthesis , RNA Nucleotidyltransferases/metabolism , RNA Stability , Synthetic Biology , Uracil Nucleotides/metabolismABSTRACT
Dcp1/2 is the major eukaryotic RNA decapping complex, comprised of the enzyme Dcp2 and activator Dcp1, which removes the 5' m(7)G cap from mRNA, committing the transcript to degradation. Dcp1/2 activity is crucial for RNA quality control and turnover, and deregulation of these processes may lead to disease development. The molecular details of Dcp1/2 catalysis remain elusive, in part because both cap substrate (m(7)GpppN) and m(7)GDP product are bound by Dcp1/2 with weak (mM) affinity. In order to find inhibitors to use in elucidating the catalytic mechanism of Dcp2, we screened a small library of synthetic m(7)G nucleotides (cap analogs) bearing modifications in the oligophosphate chain. One of the most potent cap analogs, m(7)GpSpppSm(7)G, inhibited Dcp1/2 20 times more efficiently than m(7)GpppN or m(7)GDP. NMR experiments revealed that the compound interacts with specific surfaces of both regulatory and catalytic domains of Dcp2 with submillimolar affinities. Kinetics analysis revealed that m(7)GpSpppSm(7)G is a mixed inhibitor that competes for the Dcp2 active site with micromolar affinity. m(7)GpSpppSm(7)G-capped RNA undergoes rapid decapping, suggesting that the compound may act as a tightly bound cap mimic. Our identification of the first small molecule inhibitor of Dcp2 should be instrumental in future studies aimed at understanding the structural basis of RNA decapping and may provide insight toward the development of novel therapeutically relevant decapping inhibitors.
Subject(s)
RNA Cap Analogs/chemistry , Schizosaccharomyces pombe Proteins/antagonists & inhibitors , Drug Evaluation, Preclinical , RNA Cleavage , RNA, Messenger/chemistry , Schizosaccharomyces/enzymologyABSTRACT
The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5'-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5'-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m(7)GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5'-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F.
Subject(s)
Eukaryotic Initiation Factor-4E/metabolism , Eukaryotic Initiation Factor-4G/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Protein Biosynthesis/drug effects , Protein Serine-Threonine Kinases/metabolism , RNA Caps/metabolism , RNA, Messenger/metabolism , Amino Acid Substitution , Animals , Cell-Free System/drug effects , Cell-Free System/enzymology , Cell-Free System/metabolism , Eukaryotic Initiation Factor-4E/chemistry , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-4G/chemistry , Eukaryotic Initiation Factor-4G/genetics , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Inverted Repeat Sequences , Mutant Proteins/antagonists & inhibitors , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phosphorylation/drug effects , Protein Interaction Domains and Motifs , Protein Kinase Inhibitors/pharmacology , Protein Processing, Post-Translational/drug effects , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , RNA/chemistry , RNA/metabolism , RNA Caps/chemistry , RNA Folding/drug effects , RNA, Messenger/chemistry , Rabbits , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Reticulocytes/drug effects , Reticulocytes/enzymology , Reticulocytes/metabolismABSTRACT
Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, ß- or γ-position of the 5',5'-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH3-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH3-analogs and the corresponding analogs containing S instead of BH3 (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH3-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m2 (7,2'-O)GppBH3pG D1 and m2 (7,2'-O)GppBH3pG D2, respectively, than for in vitro transcribed mRNA capped with m2 (7,3'-O)GpppG. Higher expression of cancer antigens would make mRNAs containing m2 (7,2'-O)GppBH3pG D1 and m2 (7,2'-O)GppBH3pG D2 favorable for anticancer immunization.
Subject(s)
Boranes/chemistry , Phosphates/chemistry , Protein Synthesis Inhibitors/chemistry , RNA Cap Analogs/chemistry , Animals , Caenorhabditis elegans Proteins/metabolism , Dendritic Cells/metabolism , Endoribonucleases/metabolism , Eukaryotic Initiation Factor-4E/metabolism , Humans , Neoplasms/drug therapy , Protein Biosynthesis/drug effects , Protein Synthesis Inhibitors/pharmacology , Pyrophosphatases/metabolism , RNA Cap Analogs/chemical synthesis , RNA Cap Analogs/metabolism , RNA Cap Analogs/pharmacology , StereoisomerismABSTRACT
Histone mRNAs are rapidly degraded when DNA replication is inhibited during S phase with degradation initiating with oligouridylation of the stem loop at the 3' end. We developed a customized RNA sequencing strategy to identify the 3' termini of degradation intermediates of histone mRNAs. Using this strategy, we identified two types of oligouridylated degradation intermediates: RNAs ending at different sites of the 3' side of the stem loop that resulted from initial degradation by 3'hExo and intermediates near the stop codon and within the coding region. Sequencing of polyribosomal histone mRNAs revealed that degradation initiates and proceeds 3' to 5' on translating mRNA and that many intermediates are capped. Knockdown of the exosome-associated exonuclease PM/Scl-100, but not the Dis3L2 exonuclease, slows histone mRNA degradation consistent with 3' to 5' degradation by the exosome containing PM/Scl-100. Knockdown of No-go decay factors also slowed histone mRNA degradation, suggesting a role in removing ribosomes from partially degraded mRNAs.
Subject(s)
3' Untranslated Regions , Histones/genetics , Polyribosomes/genetics , RNA Stability , Uridine/metabolism , Base Sequence , Codon , Exoribonucleases/genetics , Exoribonucleases/metabolism , Exosome Multienzyme Ribonuclease Complex/genetics , Exosome Multienzyme Ribonuclease Complex/metabolism , Gene Expression Regulation, Developmental , Gene Library , HeLa Cells , Histones/metabolism , Humans , Jurkat Cells , Molecular Sequence Data , Nucleic Acid Conformation , Open Reading Frames , Polyribosomes/metabolism , S Phase/genetics , Sequence Analysis, RNA , Signal TransductionABSTRACT
Downregulation of CPEB1, a sequence-specific RNA-binding protein, in a mouse mammary epithelial cell line (CID-9) causes epithelial-to-mesenchymal transition (EMT), based on several criteria. First, CPEB1 knockdown decreases protein levels of E-cadherin and ß-catenin but increases those of vimentin and Twist1. Second, the motility of CPEB1-depleted cells is increased. Third, CID-9 cells normally form growth-arrested, polarized and three-dimensional acini upon culture in extracellular matrix, but CPEB1-deficient CID-9 cells form nonpolarized proliferating colonies lacking a central cavity. CPEB1 downregulates Twist1 expression by binding to its mRNA, shortening its poly(A) tract and repressing its translation. CID-9 cultures contain both myoepithelial and luminal epithelial cells. CPEB1 increases during CID-9 cell differentiation, is predominantly expressed in myoepithelial cells, and its knockdown prevents expression of the myoepithelial marker p63. CPEB1 is present in proliferating subpopulations of pure luminal epithelial cells (SCp2) and myoepithelial cells (SCg6), but its depletion increases Twist1 only in SCg6 cells and fails to downregulate E-cadherin in SCp2 cells. We propose that myoepithelial cells prevent EMT by influencing the polarity and proliferation of luminal epithelial cells in a mechanism that requires translational silencing of myoepithelial Twist1 by CPEB1.
Subject(s)
Mammary Glands, Animal/cytology , Mammary Glands, Animal/metabolism , Transcription Factors/metabolism , mRNA Cleavage and Polyadenylation Factors/metabolism , Animals , Cadherins/biosynthesis , Cell Differentiation/physiology , Cell Movement/physiology , Down-Regulation , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelial-Mesenchymal Transition/physiology , Female , Gene Knockdown Techniques , HEK293 Cells , Humans , Mice , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Factors/deficiency , Transcription Factors/genetics , Twist-Related Protein 1/biosynthesis , Twist-Related Protein 1/genetics , mRNA Cleavage and Polyadenylation Factors/deficiency , mRNA Cleavage and Polyadenylation Factors/geneticsABSTRACT
We describe the synthesis and properties of five dinucleotide fluorescent cap analogues labelled at the ribose of the 7-methylguanosine moiety with either anthraniloyl (Ant) or N-methylanthraniloyl (Mant), which have been designed for the preparation of fluorescent mRNAs via transcription in vitro. Two of the analogues bear a methylene modification in the triphosphate bridge, providing resistance against either the Dcp2 or DcpS decapping enzymes. All these compounds were prepared by ZnCl2-mediated coupling of a nucleotide P-imidazolide with a fluorescently labelled mononucleotide. To evaluate the utility of these compounds for studying interactions with cap-binding proteins and cap-related cellular processes, both biological and spectroscopic features of those compounds were determined. The results indicate acceptable quantum yields of fluorescence, pH independence, environmental sensitivity, and photostability. The cap analogues are incorporated by RNA polymerase into mRNA transcripts that are efficiently translated in vitro. Transcripts containing fluorescent caps but unmodified in the triphosphate chain are hydrolysed by Dcp2 whereas those containing a α-ß methylene modification are resistant. Model studies exploiting sensitivity of Mant to changes of local environment demonstrated utility of the synthesized compounds for studying cap-related proteins.
ABSTRACT
The translational efficiency and stability of synthetic mRNA in both cultured cells and whole animals can be improved by incorporation of modified cap structures at the 5'-end. mRNAs are synthesized in vitro by a phage RNA polymerase transcribing a plasmid containing the mRNA sequence in the presence of all four NTPs plus a cap dinucleotide. Modifications in the cap dinucleotide at the 2'- or 3'-positions of m(7)Guo, or modifications in the polyphosphate chain, can improve both translational efficiency and stability of the mRNA, thereby increasing the amount and duration of protein expression. In the context of RNA-based immunotherapy, the latter is especially important for antigen production and presentation by dendritic cells. Protocols are presented for synthesis of modified mRNAs, their introduction into cells and whole animals, and measurement of their translational efficiency and stability.
Subject(s)
Protein Biosynthesis , RNA Cap Analogs/chemistry , RNA Stability , RNA, Messenger , Transfection/methods , Animals , Humans , RNA, Messenger/chemical synthesis , RNA, Messenger/chemistry , RNA, Messenger/geneticsABSTRACT
Metazoan replication-dependent histone mRNAs are only present in S-phase, due partly to changes in their stability. These mRNAs end in a unique stem-loop (SL) that is required for both translation and cell-cycle regulation. Previous studies showed that histone mRNA degradation occurs through both 5'â3' and 3'â5' processes, but the relative contributions are not known. The 3' end of histone mRNA is oligouridylated during its degradation, although it is not known whether this is an essential step. We introduced firefly luciferase reporter mRNAs containing the histone 3' UTR SL (Luc-SL) and either a normal or hDcp2-resistant cap into S-phase HeLa cells. Both mRNAs were translated, and translation initially protected the mRNAs from degradation, but there was a lag of â¼40 min with the uncleavable cap compared to â¼8 min for the normal cap before rapid decay. Knockdown of hDcp2 resulted in a similar longer lag for Luc-SL containing a normal cap, indicating that 5'â3' decay is important in this system. Inhibition of DNA replication with hydroxyurea accelerated the degradation of Luc-SL. Knockdown of terminal uridyltransferase (TUTase) 4 but not TUTase 3 slowed the decay process, but TUTase 4 knockdown had no effect on destabilization of the mRNA by hydroxyurea. Both Luc-SL and its 5' decay intermediates were oligouridylated. Preventing oligouridylation by 3'-deoxyadenosine (cordycepin) addition to the mRNA slowed degradation, in the presence or absence of hydroxyurea, suggesting oligouridylation initiates degradation. The spectrum of oligouridylated fragments suggests the 3'â5' degradation machinery stalls during initial degradation, whereupon reuridylation occurs.
Subject(s)
Oligoribonucleotides/metabolism , RNA Stability/physiology , RNA, Messenger/metabolism , Uracil Nucleotides/metabolism , 3' Untranslated Regions/physiology , DNA Replication/drug effects , Deoxyadenosines/pharmacology , Gene Silencing , HeLa Cells , Histones/metabolism , Humans , Hydroxyurea/pharmacology , Nucleic Acid Synthesis Inhibitors/pharmacology , Oligoribonucleotides/antagonists & inhibitors , Polynucleotide Adenylyltransferase , Protein Biosynthesis , RNA Stability/genetics , RNA, Messenger/chemistry , Transduction, Genetic , Uracil Nucleotides/antagonists & inhibitors , mRNA Cleavage and Polyadenylation FactorsABSTRACT
Integration of mouse mammary tumor virus (MMTV) at the common integration site Int6 occurs in the gene encoding eIF3e, the p48 subunit of translation initiation factor eIF3. Integration is at any of several introns of the Eif3e gene and causes the expression of truncated Eif3e mRNAs. Ectopic expression of the truncated eIF3e protein resulting from integration at intron 5 (3e5) induces malignant transformation, but by an unknown mechanism. Because eIF3e makes up at least part of the binding site for eIF4G, we examined the effects of 3e5 expression on protein synthesis. We developed an NIH3T3 cell line that contains a single copy of the 3e5 sequence at a predetermined genomic site. Co-immunoprecipitation indicated diminished binding of eIF3 to eIF4G, signifying a reduction in recruitment of the mRNA-unwinding machinery to the 43 S preinitiation complex. Cell growth and overall protein synthesis were decreased. Translation driven by the eIF4G-independent hepatitis C virus internal ribosome entry sequence (HCV IRES) in a bicistronic mRNA was increased relative to cap-dependent translation. Endogenous mRNAs encoding XIAP, c-Myc, CYR61, and Pim-1, which are translated in a cap-independent manner, were shifted to heavier polysomes whereas mRNAs encoding GAPDH, actin, L32, and L34, which are translated in a cap-dependent manner, were shifted to lighter polysomes. We propose that expression of 3e5 diminishes eIF4G interaction with eIF3 and causes abnormal gene expression at the translational level. The correlation between up-regulation of cap-independent translation and MMTV-induced tumorigenesis contrasts with the well established model for malignant transformation involving up-regulation of highly cap-dependent translation.
Subject(s)
Eukaryotic Initiation Factor-3/genetics , Eukaryotic Initiation Factor-4G/metabolism , Mammary Tumor Virus, Mouse , Protein Biosynthesis , RNA Caps/genetics , Virus Integration/genetics , Animals , Cell Transformation, Neoplastic , Eukaryotic Initiation Factor-3/metabolism , Gene Expression , Introns , Mice , NIH 3T3 Cells , Polyribosomes , Protein Subunits , RNA, MessengerABSTRACT
Helicobacter pylori persistently colonizes humans, causing gastritis, ulcers, and gastric cancer. Adherence to the gastric epithelium has been shown to enhance inflammation, yet only a few H. pylori adhesins have been paired with targets in host tissue. The alpAB locus has been reported to encode adhesins involved in adherence to human gastric tissue. We report that abrogation of H. pylori AlpA and AlpB reduces binding of H. pylori to laminin while expression of plasmid-borne alpA or alpB confers laminin-binding ability to Escherichia coli. An H. pylori strain lacking only AlpB is also deficient in laminin binding. Thus, we conclude that both AlpA and AlpB contribute to H. pylori laminin binding. Contrary to expectations, the H. pylori SS1 mutant deficient in AlpA and AlpB causes more severe inflammation than the isogenic wild-type strain in gerbils. Identification of laminin as the target of AlpA and AlpB will facilitate future investigations of host-pathogen interactions occurring during H. pylori infection.
Subject(s)
Adhesins, Bacterial/metabolism , Bacterial Adhesion , Bacterial Outer Membrane Proteins/metabolism , Helicobacter Infections/pathology , Helicobacter pylori/pathogenicity , Host-Pathogen Interactions , Laminin/metabolism , Animals , Escherichia coli/genetics , Female , Gastric Mucosa/microbiology , Gastric Mucosa/pathology , Gene Expression , Gerbillinae , Helicobacter Infections/microbiology , Inflammation/pathology , Male , PlasmidsABSTRACT
Decapping is an essential step in multiple pathways of mRNA degradation. Previously, we synthesized mRNAs containing caps that were resistant to decapping, both to dissect the various pathways for mRNA degradation and to stabilize mRNA for more sustained protein expression. mRNAs containing an α-ß CH(2) group are resistant to in vitro cleavage by the decapping enzyme hDcp2 but poorly translated. mRNAs containing an S substitution at the ß-phosphate are well translated but only partially resistant to hDcp2. We now describe seven new cap analogs substituted at the ß-phosphate with BH(3) or Se, or substituted at either the α-ß or ß-γ O with NH. The analogs differ in affinity for eIF4E and efficiency of in vitro incorporation into mRNA by T7 RNA polymerase. Luciferase mRNAs capped with these analogs differ in resistance to hDcp2 hydrolysis in vitro, translational efficiency in rabbit reticulocyte lysate and in HeLa cells, and stability in HeLa cells. Whereas mRNAs capped with m(2)(7,2'-O)Gpp(S)pG were previously found to have the most favorable properties of translational efficiency and stability in mammalian cells, mRNAs capped with m(7)Gpp(BH3)pm(7)G are translated with the same efficiency but are more stable. Interestingly, some mRNAs exhibit a lag of up to 60 min before undergoing first-order decay (t(1/2) â 25 min). Only mRNAs that are efficiently capped, resistant to decapping in vitro, and actively translated have long lag phases.
Subject(s)
Boric Acids/metabolism , Nitrogen Compounds/metabolism , Polyphosphates/metabolism , Protein Biosynthesis , RNA Stability , RNA, Messenger/analysis , Selenium/metabolism , Animals , DNA-Directed RNA Polymerases/metabolism , Endoribonucleases/metabolism , HeLa Cells , Humans , Mice , Molecular Structure , Polyphosphates/chemistry , RNA Caps , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Rabbits , Reticulocytes/chemistry , Stereoisomerism , Substrate Specificity , Viral Proteins/metabolismABSTRACT
It was previously shown that integrin α6ß4 contributes to translation of cancer-related mRNAs such as VEGF via initiation factor eIF4E. In this study, we found that integrin α6ß4 regulates the activity of eIF4E through the Ser/Thr kinase Mnk. Although a role for Mnk in various aspects of cancer progression has been established, a link between integrin and Mnk activity has not. Here we show that Mnk1 is a downstream effector of integrin α6ß4 and mediates the α6ß4 signaling, important for translational control. Integrin α6ß4 signals through MEK and p38 MAPK to increase phosphorylation of Mnk1 and eIF4E. Inhibition of Mnk1 activity by CGP57380 or downregulation by shRNA blocks α6ß4-dependent translation of VEGF mRNA. Our studies suggest that Mnk1 could be a therapeutic target in cancers where the integrin α6ß4 level is high.
Subject(s)
Eukaryotic Initiation Factor-4E/metabolism , Integrin alpha6beta4/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Vascular Endothelial Growth Factor A/genetics , Aniline Compounds/pharmacology , Butadienes/pharmacology , Cell Growth Processes/drug effects , Cell Line, Tumor , Culture Media, Serum-Free , Down-Regulation , Eukaryotic Initiation Factor-4E/genetics , Humans , Integrin alpha6beta4/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , MAP Kinase Kinase Kinases/antagonists & inhibitors , MAP Kinase Kinase Kinases/metabolism , Nitriles/pharmacology , Phosphorylation , Protein Biosynthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Purines/pharmacology , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/biosynthesis , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
We describe the chemical synthesis and preliminary biophysical and biochemical characterization of a series of mRNA 5' end (cap) analogs designed as reagents for obtaining mRNA molecules with augmented translation efficiency and stability in vivo and as useful tools to study mRNA metabolism. The analogs share three structural features: (i) 5',5'- bridge elongated to tetraphosphate to increase their affinity to translation initiation factor eIF4E (ii) a single phosphorothioate modification at either the α, ß, γ or δ-position of the tetraphosphate to decrease their susceptibility to enzymatic degradation and/or to modulate their interaction with specific proteins and (iii) a 2'-O-methyl group in the ribose of 7-methylguanosine, characteristic to Anti-Reverse Cap Analogs (ARCAs), which are incorporated into mRNA during in vitro transcription exclusively in the correct orientation. The dinucleotides bearing modified tetraphosphate bridge were synthesized by ZnCl(2) mediated coupling between two mononucleotide subunits with isolated yields of 30-65%. The preliminary biochemical results show that mRNAs capped with new analogs are 2.5-4.5 more efficiently translated in a cell free system than m(7)GpppG-capped mRNAs, which makes them promising candidates for RNA-based therapeutic applications such as gene therapy and anti-cancer vaccines.
ABSTRACT
Caenorhabditis elegans expresses five family members of the translation initiation factor eIF4E whose individual physiological roles are only partially understood. We report a specific role for IFE-2 in a conserved temperature-sensitive meiotic process. ife-2 deletion mutants have severe temperature-sensitive chromosome-segregation defects. Mutant germ cells contain the normal six bivalents at diakinesis at 20 degrees C but 12 univalents at 25 degrees C, indicating a defect in crossover formation. Analysis of chromosome pairing in ife-2 mutants at the permissive and restrictive temperatures reveals no defects. The presence of RAD-51-marked early recombination intermediates and 12 well condensed univalents indicate that IFE-2 is not essential for formation of meiotic double-strand breaks or their repair through homologous recombination but is required for crossover formation. However, RAD-51 foci in ife-2 mutants persist into inappropriately late stages of meiotic prophase at 25 degrees C, similar to mutants defective in MSH-4/HIM-14 and MSH-5, which stabilize a critical intermediate in crossover formation. In wild-type worms, mRNAs for msh-4/him-14 and msh-5 shift from free messenger ribonucleoproteins to polysomes at 25 degrees C but not in ife-2 mutants, suggesting that IFE-2 translationally upregulates synthesis of MSH-4/HIM-14 and MSH-5 at elevated temperatures to stabilize Holliday junctions. This is confirmed by an IFE-2-dependent increase in MSH-5 protein levels.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , DNA-Binding Proteins/metabolism , Eukaryotic Initiation Factor-4E/metabolism , Meiosis/physiology , Protein Biosynthesis , RNA, Messenger/metabolism , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Crossing Over, Genetic , DNA/genetics , DNA/metabolism , DNA/radiation effects , DNA Breaks, Double-Stranded , DNA-Binding Proteins/genetics , Eukaryotic Initiation Factor-4E/genetics , Female , Hot Temperature , Male , Mutation , Oogenesis/physiology , Phenotype , RNA, Messenger/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Spermatogenesis/physiologyABSTRACT
Eukaryotic initiation factor 4E (eIF4E) has long been known as the cap-binding protein that participates in recruitment of mRNA to the ribosome. A number of recent advances have not only increased our understanding of how eIF4E acts in translation but also uncovered non-translational roles. New structures have been determined for eIF4E in complex with various ligands and for other cap-binding proteins. We have also learned that most eukaryotic organisms express multiple eIF4E family members, some involved in general translation but others having specialized functions, including repression of translation. A number of new eIF4E-binding proteins have been reported, some of which tether it to specific mRNAs.
Subject(s)
Eukaryotic Initiation Factor-4E/metabolism , Aging , Amino Acid Sequence , Animals , Binding Sites/genetics , Body Patterning , Eukaryotic Initiation Factor-4E/genetics , Female , Humans , Male , Models, Biological , Oogenesis , Oviposition , Protein Binding , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolism , SpermatogenesisABSTRACT
To investigate the binding specificity of turnip mosaic virus (TuMV) viral protein-genome linked (VPg) with translation initiation factor 4E, we evaluated here the kinetic parameters for the interactions of human eIF4E, Caenorhabditis elegans IFE-3 and IFE-5 and Arabidopsis eIFiso4E, by surface plasmon resonance (SPR). The results indicated that TuMV VPg does not show a binding preference for Arabidopsis eIFiso4E, even though it is from a host species whereas the other eIF4E orthologues are not. Surprisingly, the effect of m(7)GTP on both the rate constants and equilibrium binding constants for the interactions of VPg differed for the four eIF4E orthologues. In the case of eIFiso4E and IFE-3, m(7)GTP increased k(on), but for eIF4E and IFE-5, it decreased k(on). To provide insight into the structural basis for these differences in VPg binding, tertiary structures of the eIF4E orthologues were predicted on the basis of the previously determined crystal structure of m(7)GpppA-bound human eIF4E. The results suggested that in cap-bound eIF4E orthologues, the VPg binds to the C-terminal region, which constitutes one side of the entrance to the cap-binding pocket, whereas in the cap-free state, VPg binds to the widely opened cap-binding pocket and its surrounding region. The binding of VPg to the C-terminal region was confirmed by the SPR analyses of N- or C-terminal residues-deleted eIF4E orthologues.
Subject(s)
Eukaryotic Initiation Factor-4E/chemistry , Eukaryotic Initiation Factor-4E/metabolism , Genome, Viral/genetics , Host-Pathogen Interactions , Plant Viruses/genetics , RNA Caps/metabolism , Viral Proteins/metabolism , Amino Acid Sequence , Animals , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Humans , Immobilized Proteins/metabolism , Kinetics , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity , Surface Plasmon ResonanceABSTRACT
Interaction of the mRNA cap with the translational machinery is a critical and early step in the initiation of protein synthesis. To better understand this process, we determined kinetic constants for the interaction of m(7)GpppG with human eIF4E by stopped-flow fluorescence quenching in the presence of a 90-amino acid fragment of human eIF4G that contains the eIF4E-binding domain (eIF4G(557-646)). The values obtained, k(on) = 179 x 10(6) m(-1) s(-1) and k(off) = 79 s(-1), were the same as reported previously in the absence of an eIF4G-derived peptide. We also used surface plasmon resonance to determine kinetic constants for the binding of eIF4E to eIF4G(557-646), both in the presence and absence of m(7)GpppG. The results indicated that eIF4G(557-646) binds eIF4E and eIF4E.m(7)GpppG at the same rate, with k(on) = 3 x 10(6) m(-1) s(-1) and k(off) = 0.01 s(-1). Our data represent the first full kinetic description of the interaction of eIF4E with its two specific ligands. The results demonstrate that the formation of the m(7)GpppG.eIF4E.eIF4G(557-646) complex obeys a sequential, random kinetic mechanism and that there is no preferential pathway for its formation. Thus, even though eIF4G(557-646) binds eIF4E tightly, it does not increase the affinity of eIF4E for m(7)GpppG, as has been claimed in several previous publications. We did, in fact, observe increased binding to m(7)GTP-Sepharose in the presence of eIF4G(557-646), but only with recombinant eIF4E that was prepared from inclusion bodies.