Genetic regulation of translatable mRNA levels for mouse sn-glycerol-3-phosphate dehydrogenase during development of the cerebellum.
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Animals↗
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Results for “RNA Cap Analogs”
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Characterization of a protein synthesis system from rat liver. Translation of endogenous and exogenous messenger RNA.
An in vitro system prepared from rat liver postmitochondrial supernatant exhibits a high rate of protein synthesis for an extended period of time. This system initiates translation of either endogenous or exogenous mRNA, incorporates Met at a rate of 13 pmol/mg of postmitochondrial supernatant protein/min, maintains this rate for at least 90 min, and performs several rounds of translation/mRNA molecule. Up to 50% of the activity is due to reinitiation of protein synthesis using endogenous mRNA. In addition, 60-70% of the protein synthesized was released from ribosomes into the medium. Addition of globin mRNA stimulates protein synthesis and results in the synthesis of a protein that comigrates with authentic rabbit globin. Black beetle virus mRNA 2 also stimulates protein synthesis and results in synthesis of a protein with molecular weight corresponding to that of the mature viral protein. With endogenous rat liver mRNA this system synthesizes a large number of proteins.
Amino Acids↗
Overexpression of initiation factor eIF-4E does not relieve the translational repression of ribosomal protein mRNAs in quiescent cells.
Translation of ribosomal protein (rp) mRNA is selectively repressed in mouse erythroleukemia (MEL) cells, which cease to proliferate upon differentiation, and in NIH 3T3 cells, for which growth is arrested by either serum starvation, contact inhibition, or treatment with the DNA polymerase inhibitor, aphidicolin. The efficiency of translation of rp mRNAs correlates with the expression of the gene encoding the cap binding protein, eIF-4E, as indicated by the fact that the abundance of the corresponding mRNA and protein also fluctuates in a growth-dependent manner. To examine the hypothesis that eIF-4E plays a role in regulation of the translation efficiency of rp mRNAs, we utilized an NIH 3T3-derived eIF-4E-overexpressing cell line. These cells overproduce eIF-4E to the extent that even under conditions of growth arrest, the abundance of the respective protein in its active (phosphorylated) form is higher than that found in exponentially growing NIH 3T3 cells. Nevertheless, this surplus amount of eIF-4E does not prevent the translational repression of rp mRNAs when the growth of these cells is arrested by blocking DNA synthesis with aphidicolin or hydroxyurea. In complementary experiments we used an in vitro translation system to compare the competitive potential of mRNAs, containing the translational cis-regulatory element (5' terminal oligopyrimidne tract) and mRNAs lacking such a motif, for the cap binding protein. Our results demonstrate that both types of mRNAs, regardless of their translational response to growth arrest, exhibit similar sensitivity to the cap analogue m7G(5')ppp(5')G. It appears, therefore, that the presence of the regulatory sequence at the 5' terminus of rp mRNAs does not lessen its competitive potential for the cap binding protein and that the growth-dependent decrease in the activity of eIF-4E does not play a key role in the repression of translation of rp mRNAs.
3T3 Cells↗
In vitro synthesis of human protein synthesis initiation factor 4 gamma and its localization on 43 and 48 S initiation complexes.
The rate of protein synthesis is controlled in a large number of physiological situations at the stage of 48 S initiation complex formation, a phase that involves the recruitment of mRNA to the 40 S ribosomal subunit. This process is mediated by the eukaryotic initiation factor-4 (eIF-4) group of translation initiation factors consisting of eIF-4E, eIF-4A, eIF-4B, and eIF-4 gamma. In order to develop a new tool to study this process, we have produced radiolabeled eIF-4 gamma by in vitro transcription and translation. Despite the fact that eIF-4 gamma is predicted from the cDNA sequence to be 154 kDa, the major synthetic product migrated on SDS-polyacrylamide gel electrophoresis at 205 kDa. Although this is similar to the migration of the fastest polypeptide of authentic eIF-4 gamma (approximately 206 kDa), no products were found to co-migrate with the slowest forms of authentic eIF-4 gamma (210-220 kDa), suggesting that these forms derive from extensive modification of the initial polypeptide. The in vitro product also formed a complex with eIF-4E, as judged by its ability to bind to m7GTP-Sepharose. Sucrose gradient sedimentation studies demonstrated that eIF-4 gamma was present on both 43 and 48 S initiation complexes but not 80 S complexes. This supports a model in which free eIF-4E binds to mRNA followed by binding of the eIF-4E.mRNA complex to a 43 S initiation complex already containing eIF-4 gamma.
Cell-Free System↗
Unique features of internal initiation of hepatitis C virus RNA translation.
The question of whether hepatitis C virus (HCV) RNA is translated by a mechanism of internal ribosome entry has been examined by testing whether insertion of HCV sequences between the two cistrons of a dicistronic mRNA promotes translation of the downstream cistron in rabbit reticulocyte lysates. Deletion analysis showed that efficient internal initiation required a segment of the HCV genome extending from about nucleotides 40-370 and that deletions from the 3'-end of this element were highly deleterious. As the authentic initiation codon for HCV polyprotein synthesis is at nucleotide 342, this demonstrates that, besides 5'-UTR sequences, a short length of HCV coding sequences is required for internal initiation. This finding was confirmed in transfection assays of BT7-H cells and was shown to be independent of the nature of the downstream reporter cistron. The strong requirement for coding sequences is in sharp contrast to internal initiation of picornavirus RNA translation. As a probable correlate with this, it was also found that the efficiency of internal initiation was only marginally compromised when the authentic initiation codon was mutated to a non-AUG codon, again in sharp contrast with the picornaviruses. The finding that coding sequences are required for internal initiation has important implications for the design of experiments to test for internal initiation of translation of cellular mRNAs.
Animals↗
Evidence that the genomic RNA of hepatitis E virus is capped.
Hepatitis E virus (HEV) is an unclassified virus with a positive-sense RNA genome and an undefined replication strategy. In order to determine whether the HEV genome is capped or not, we developed a reverse transcription-PCR assay that is based on the ability of a monoclonal antibody to recognize 7-methylguanosine (m(7)G). Antibody to m(7)G bound RNA extracted from virions of two different HEV genotypes. The cap analog competitively inhibited the binding of virion RNAs, demonstrating that HEV has a capped RNA genome.
Genome, Viral↗
Additional methylation at the N(2)-position of the cap of 26S Semliki Forest virus late mRNA and initiation of translation.
Cap analysis of the late 26S Semliki Forest viral mRNA reveals that almost 30% of the caps possess both a methyl group at the N(7)-position and one at the N(2)-position. We have compared the degree of methylation of the caps of polysomal and non-polysomal 26S mRNA in order to check whether this feature is responsible for its translation late in infection. It was found that extra methyl groups on the caps cause a lower rate of initiation. Polysomal 26S mRNA contained less m2,7G- and m2,2,7G-caps than free 26S. The cap analog m2,2,7Gp was slightly less inhibitory than m7Gp in an in vitro translation system.
Methylation↗
Novel cap analogs for in vitro synthesis of mRNAs with high translational efficiency.
Synthetic analogs of the N7-methylated guanosine triphosphate cap at the 5' end of eukaryotic mRNAs and snRNAs have played an important role in understanding their splicing, intracellular transport, translation, and turnover. We report here a new series of N7-benzylated dinucleoside tetraphosphate analogs, b7Gp4G, b7m(3'-O)Gp4G, and b7m2Gp4G, that extend our knowledge of the role of the cap in translation. We used these novel analogs, along with 10 previously synthesized analogs, to explore five parameters: binding affinity to eIF4E, inhibition of cap-dependent translation in a rabbit reticulocyte lysate system, efficiency of incorporation into RNAs during in vitro transcription (% capping), orientation of the analog in the synthetic mRNA (% correct orientation), and in vitro translational efficiency of mRNAs capped with the analog. The 13 cap analogs differed in modifications of the first (distal) and second (proximal) guanine moieties, the first and second ribose moieties, and the number of phosphate residues. Among these were analogs of the naturally occurring cap m3(2,2,7)Gp3G. These compounds varied by 61-fold in affinity for eIF4E, 146-fold in inhibition of cap-dependent translation, 1.4-fold in % capping, and 5.6-fold in % correct orientation. The most stimulatory analog enhanced translation 44-fold compared with uncapped RNA. mRNAs capped with b7m2Gp4G, m7Gp3m7G, b7m(3'-OGp4G, and m7Gp4m7G were translated 2.5-, 2.6-, 2.8-, and 3.1-fold more efficiently than mRNAs capped with m7Gp3G, respectively. Relative translational efficiencies could generally be explained in terms of cap affinity for eIF4E, % capping, and % correct orientation. The measurement of all five parameters provides insight into factors that contribute to translational efficiency.
Animals↗
Synthesis and properties of mRNA cap analogs containing phosphorothioate moiety in 5',5'-triphosphate chain.
Nucleosides and oligonucleotides with an oxygen replaced by sulfur atom are an interesting class of compounds because of their improved stability toward enzymatic cleavage by nucleases. We have synthesized several dinucleotide mRNA cap analogs containing a phosphorothioate moiety in the alpha, beta, or gamma position of 5',5'-triphosphate chain [m7Gp(s)ppG, m7Gpp(s)pG, and m7Gppp(s)G]. These are the first examples of the biologically important 5'mRNA cap analogs containing a phosphorothioate moiety, and these compounds may be useful in a variety of biochemical and biotechnological applications. Incorporation of a sulfur atom in the alpha or gamma position within the dinucleotide cap analog was achieved using PSCl3 in a nucleoside phosphorylation reaction followed by coupling the phosphorothioate of nucleoside with a second nucleotide. Synthesis of cap analogs with the phosphorothioate moiety in beta position was performed using an organic phosphorothioate salt in a coupling reaction with an activated nucleotide. The structures of newly synthesized compounds was confirmed using MS and 1H and 31P NMR spectroscopy. We present here the results of preliminary studies on their interaction with translation initiation factor eIF4E and enzymatic hydrolysis with human and nematode DcpS scavengers.
Animals↗
Guanosine nucleotide analogs as inhibitors of alphavirus mRNA capping enzyme.
The two virus-specific reactions in the capping of alphavirus RNAs, catalyzed by the replicase protein nsP1, are promising targets for developing virus-specific inhibitors. In this report, we have studied the effect of over 50 cap analogs on the guanine-7-methyltransferase and guanylyltransferase activities of Semliki Forest virus nsP1. Recombinant nsP1 was expressed in Escherichia coli and partially purified by flotation in a discontinuous sucrose gradient. The methyltransferase activity had a pH optimum between pH 6.5 and 7.1, and the apparent Km values were 1.9 mM for GTP, 6.0 microM for S-adenosyl-L-methionine and 170 microM for Mg2+. NsP1 methyltransferase was able to methylate efficiently GTP (relative activity 100%), GDP (16%), GpppG (35%), GppppG (50%) and less efficiently GpppA (12%), m2GTP (9%), and m2,2GTP (25%), but not m7GppG. The most potent inhibitors for nsP1 methyltransferase were et2m7GMP (Ki value 42 microM), m2,7GMP, (64 microM), m2,7GpppG (82 microM), m2et7GMP (105 microM), m2(2-phet)7GMP (194 microM) and m2GMP (386 microM). Of these compounds, m2GMP, m2et7GMP and m2(2-phet)7GMP showed competitive inhibition, whereas the others showed mixed type inhibition. All compounds that inhibited the methyltransferase activity inhibited also the guanylyltransferase activity of nsP1.
Enzyme Inhibitors↗
Wheat germ poly(A) binding protein enhances the binding affinity of eukaryotic initiation factor 4F and (iso)4F for cap analogues.
Most eukaryotic mRNAs contain a 5' cap (m7GppX) and a 3' poly(A) tail to increase synergistically the translational efficiency. Recently, the poly(A) binding protein (PABP) and cap-binding protein, eIF-4F, were found to interact [Le et al. (1997) J. Biol. Chem. 272, 16247-16255; Tarun and Sachs (1996) EMBO J. 15, 7168-7177]. These data suggest that PABP may exert its effect on translational efficiency either by increasing the formation of initiation factor-mRNA complex or by enhancing ribosome recycling. To investigate the functional consequences of these interactions, the fluorescent cap analogue, ant-m7GTP, which is an environmentally sensitive fluorescent probe [Ren and Goss (1996) Nucleic Acids Res. 24, 3629-3634] was used to investigate the cap-binding affinity. Our data show that the binding of eIF-(iso)4F or eIF-4F to cap analogue enhanced their binding affinity toward PABP approximately 40-fold. Similarly, the eIF-4F/PABP or eIF-(iso)4F/PABP complexes show a 40-fold enhancement of cap analogue binding as compared to eIF-4F or eIF-(iso)4F alone. At least part of the enhancement of the translational initiation by PABP can be accounted for by direct changes in cap-binding affinity. The interactions of these components also suggest a mechanism whereby the poly(A) tail is brought into close proximity with m7G cap. This effect was examined by fluorescence energy transfer, and it was determined that the PABP/eIF-4F complex could bind both poly(A) and 5' cap simultaneously.
Binding Sites↗
A nuclear cap binding protein complex involved in pre-mRNA splicing.
A cap-binding protein complex (CBC) present in the nuclei of HeLa cells has been characterized. Purified CBC consists of two previously identified proteins, CBP80 and CBP20. These proteins are shown to cofractionate to apparent homogeneity and to be coimmunoprecipitable with anti-CBP80 antibodies. Analysis of the inhibition of pre-mRNA splicing in vitro and in vivo by chemically modified analogs of the cap structure, and of the binding of these analogs to CBC in vitro, suggests a role for the complex in splicing. Extracts immunodepleted of CBC do not efficiently splice an adenoviral pre-mRNA owing to blockage of an early step in splicing complex formation. CBC may therefore play a role in pre-mRNA recognition.
Amino Acid Sequence↗
Nonspecific effect of m7GMP on protein-RNA interactions.
Cap analogs m7GMP and m7GDP inhibit binding of eukaryotic initiation factors to reovirus capped mRNA but also inhibit complex formation involving uncapped mRNA or 18 S rRNA. Furthermore, Escherichia coli DNA-dependent RNA polymerase binds 18 S rRNA and this interaction is also blocked by m7GMP. These results indicate that inhibition by cap analogs is not a stringent test for putative cap-specific binding between proteins and mRNA.
DNA-Directed RNA Polymerases↗
Effects of pH on kinetics of binding of mRNA-cap analogs by translation initiation factor eIF4E.
Stopped-flow spectrofluorimetry and a theoretical method for predicting protonation equilibria in polyelectrolytes were combined in an analysis of the pH dependence of the kinetics of binding of analogues of the 5'-mRNA cap to the cap binding protein eIF4E. The computer simulations and available experimental data indicate that there are two titratable groups in the binding site of the protein and two titratable groups on the ligands directly involved in the binding, in addition to stacking interactions described by other groups. The observed pH dependencies of the rate constants obtained from the stopped-flow experiments are consistent with this finding. In particular, it is concluded that binding of both forms of the cap analogs regarding protonation at the N1 position of the guanine ring is efficient, and the shift to a predominantly protonated form of the ring takes place after formation of the complex.
Animals↗
An industrial process for selective synthesis of 7-methyl guanosine 5'-diphosphate: versatile synthon for synthesis of mRNA cap analogues.
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.
Guanosine Diphosphate↗
Virus-specific mRNA capping enzyme encoded by hepatitis E virus.
Hepatitis E virus (HEV), a positive-strand RNA virus, is an important causative agent of waterborne hepatitis. Expression of cDNA (encoding amino acids 1 to 979 of HEV nonstructural open reading frame 1) in insect cells resulted in synthesis of a 110-kDa protein (P110), a fraction of which was proteolytically processed to an 80-kDa protein. P110 was tightly bound to cytoplasmic membranes, from which it could be released by detergents. Immunopurified P110 catalyzed transfer of a methyl group from S-adenosylmethionine (AdoMet) to GTP and GDP to yield m(7)GTP or m(7)GDP. GMP, GpppG, and GpppA were poor substrates for the P110 methyltransferase. There was no evidence for further methylation of m(7)GTP when it was used as a substrate for the methyltransferase. P110 was also a guanylyltransferase, which formed a covalent complex, P110-m(7)GMP, in the presence of AdoMet and GTP, because radioactivity from both [alpha-(32)P]GTP and [(3)H-methyl]AdoMet was found in the covalent guanylate complex. Since both methyltransferase and guanylyltransferase reactions are strictly virus specific, they should offer optimal targets for development of antiviral drugs. Cap analogs such as m(7)GTP, m(7)GDP, et(2)m(7)GMP, and m(2)et(7)GMP inhibited the methyltransferase reaction. HEV P110 capping enzyme has similar properties to the methyltransferase and guanylyltransferase of alphavirus nsP1, tobacco mosaic virus P126, brome mosaic virus replicase protein 1a, and bamboo mosaic virus (a potexvirus) nonstructural protein, indicating there is a common evolutionary origin of these distantly related plant and animal virus families.
Cell Line↗
Novel "anti-reverse" cap analogs with superior translational properties.
Synthetic analogs of the 5'-terminal caps of eukaryotic mRNAs and snRNAs are used in elucidating such physiological processes as mRNA translation, pre-mRNA splicing, intracellular transport of mRNA and snRNAs, and mRNA turnover. Particularly useful are RNAs capped with synthetic analogs, which are produced by in vitro transcription of a DNA template using a bacteriophage RNA polymerase in the presence of ribonucleoside triphosphates and a cap dinucleotide such as m(7)Gp(3)G. Unfortunately, because of the presence of a 3'-OH on both the m(7)Guo and Guo moieties, up to half of the mRNAs contain caps incorporated in the reverse orientation. Previously we designed and synthesized two "anti-reverse" cap analogs (ARCAs), m(7)3'dGp(3)G and m(2)(7,3'-)(O)Gp(3)G, that cannot be incorporated in the reverse orientation because of modifications at the C3' position of m(7)Guo. In the present study, we have synthesized seven new cap analogs modified in the C2' and C3' positions of m(7)Guo and in the number of phosphate residues, m(2)(7,2'-)(O)Gp(3)G, m(7)2'dGp(3)G, m(7)2'dGp(4)G, m(2)(7,2'-)(O)Gp(4)G, m(2)(7,3'-)(O)Gp(4)G, m(7)Gp(5)G, and m(2)(7,3'-)(O)Gp(5)G. These were analyzed for conformation in solution, binding affinity to eIF4E, inhibition of in vitro translation, degree of reverse capping during in vitro transcription, capping efficiency, and the ability to stimulate cap-dependent translation in vitro when incorporated into mRNA. The results indicate that modifications at C2', like those at C3', prevent reverse incorporation, that tetra- and pentaphosphate cap analogs bind eIF4E and inhibit translation more strongly than their triphosphate counterparts, and that tetraphosphate ARCAs promote cap-dependent translation more effectively than previous cap analogs.
Animals↗
Multiple cis-acting signals for export of pre-U1 snRNA from the nucleus.
We have identified cis-acting sequences that promote nuclear export of pre-U1 RNA injected into Xenopus oocyte nuclei. At least three elements, the 5' m7G cap, the 3'-terminal stem-loop structure, and sequences in the 5'-terminal 124 nucleotides, contribute to efficient export of this RNA. Both the 5' and 3' export signals can function separately and do so independently of the cap structure. Experiments using hybrid RNAs indicate that the 5' and 3' export sequences of U1 RNA are sufficient to direct export of the heterologous, otherwise nonexportable, U6 RNA. The absence of comparable export signals in U6 RNA appears to be responsible for its retention in the nucleus. Stability of the pre-snRNAs in the nucleus depends on the presence of both a 5' cap structure and a 3' base-paired stem. The 5' m7G cap is neither sufficient nor necessary for nuclear export. The m7G cap by itself did not promote export of U6 RNA or nonspecific small RNAs. Moreover, substitution of this cap with either an AppG cap or gamma-mppG cap did not eliminate export of either full-length or a "minimal" U1 RNA (lacking most of the internal U1 RNA sequences), but it reduced the rate of export by about two to threefold. However, in the absence of the 3' stem-loop, substitution of the m7G cap led to a greater decrease in export rate, underscoring the cooperative action of the three different export elements of pre-U1 RNA. The m7G cap analog, m7GpppG, selectively destabilized pre-U1 RNA within the nucleus. Thus, nuclear components that recognize the 5' m7G cap may be important for both the stability and the export of pre-U1 RNA.
Animals↗