Synthesis of long, capped transcripts in vitro by SP6 and T7 RNA polymerases.
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DNA Restriction Enzymes↗
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The mRNA assembly line: transcription and processing machines in the same factory.
Processing of RNA precursors to their mature form often occurs co-transcriptionally. Consequently, the ternary complex of DNA template, RNA polymerase and nascent RNA chain is the physiological substrate for factors that modify the nascent RNA by capping, splicing and cleavage/polyadenylation. mRNA production is thought to occur within a "factory" that contains the RNA polymerase II transcription machine and the processing machines. Newly discovered protein-protein contacts between RNA polymerase and factors that process mRNA precursors are beginning to illuminate how the "mRNA factory" works.
3' Untranslated Regions↗
Immunospecific binding of capped mRNA of reovirus by an antibody to a cap structures; m7G(5')pppGm.
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Animals↗
Functional characterization of eukaryotic mRNA cap binding protein complex: effects on translation of capped and naturally uncapped RNAs.
We examined the effects of a eukaryotic mRNA cap binding protein (CBP) complex purified by cap analogue affinity chromatography [Edery, I., Humebelin, M., Darveau, A., Lee, K.A. W., Milburn, S., Hershey, J.W.B., Trachsel, H., & Sonenberg, N. (1983) J. Biol. Chem. 258, 11398 11403], on translation of several capped and naturally uncapped mRNAs in extracts prepared from poliovirus-infected or mock-infected HeLa cells. The CBP complex has activity that restores capped mRNA (globin, tobacco mosaic virus, and others) function in extracts from poliovirus-infected HeLa cells. Translation of two naturally uncapped RNAs (poliovirus and mengovirus RNAs), the translation of which is not restricted in extracts from poliovirus-infected cells, is also not stimulated by the CBP complex. Translation of several capped eukaryotic mRNAs (vesicular stomatitis virus, reovirus, and tobacco mosaic virus) in extracts from mock-infected cells is inhibited when the potassium ion concentration is increased. However, translation of capped AMV-4 RNA, which has negligible secondary structure at its 5' end, is resistant to this inhibition. Furthermore, the CBP complex reverses the high salt induced inhibition of translation of the former mRNAs. Since mRNA secondary structure is more stable at elevated salt concentrations, these data are consistent with a model in which the CBP complex has a role in melting mRNA secondary structure involving 5'-proximal sequences, to facilitate ribosome binding.
Avian Myeloblastosis Virus↗
Isolation and characterization of Dcp1p, the yeast mRNA decapping enzyme.
A major mechanism of mRNA decay occurs by the process of deadenylation, decapping and 5' --> 3' exonucleolytic degradation. Recently, the product of the DCP1 gene has been shown to be required for decapping mRNAs in vivo and co-purifies with decapping activity in vitro. We have purified Dcp1p to homogeneity and shown that it is sufficient for decapping, thereby indicating that Dcp1p is the decapping enzyme. Characterization of Dcp1p activity in vitro indicated that the 7-methyl group of the cap structure contributes to the enzyme's substrate specificity. In addition, Dcp1p was effectively inhibited by uncapped mRNAs, and the enzyme efficiently cleaved substrates that were >/=25 nucleotides in length, with a preference for longer mRNA substrates. These properties suggest that Dcp1p recognizes the mRNA substrate by interactions with both the cap and the RNA moiety. The Dcp1p is also a phosphoprotein, suggesting its activity may be regulated by post-transcriptional modification.
Endoribonucleases↗
Guanosine 5'-O-(3-thiotriphosphate) inhibits capping of reovirus mRNA.
RNA synthesized by reovirus cores was not capped or methylated when GTP was replaced by guanosine 5'-O-(3-thiotriphosphate) [gamma-S]GTP as a reaction substrate. Full length transcripts were synthesized in the presence of [gamma-S]GTP which indicated that capping could be dissociated from the process of transcription. Exogenous [gamma-S]GTP was not cleaved by the virion-associated phosphohydrolase and the majority of transcripts contained triphosphorylated 5'-termini. [gamma-S]GTP did, however, act as a donor for the capping of RNA chains initiated with GTP. Chromatography on mercury-agarose of products initiated with [gamma-S]GTP resulted in the binding of only the small (14 S) size class of RNA, although transcripts not bound to the affinity column were also thiophosphorylated.
Guanosine 5'-O-(3-Thiotriphosphate)↗
Barley yellow dwarf virus RNA requires a cap-independent translation sequence because it lacks a 5' cap.
A 3' translation enhancer (3'TE) sequence that facilitates cap-independent translation is located near the 3' end of barley yellow dwarf luteovirus RNA. Here, we show that the 3'TE is required for translation of the viral genome and thus for viral replication. Antisense inhibition showed that the 3'TE has significant secondary structure and is required for translation of virion RNA from infected plants and uncapped genomic transcripts but not for translation of capped transcripts. Direct end-labeling of RNAs verified the absence of a 5' modification on virion RNA. Thus barley yellow dwarf virus differs from related viruses by having neither a genome-linked protein nor a 5' cap.
Enhancer Elements, Genetic↗
The nucleotide sequence and genome organization of the RNA-1 segment in two bromoviruses: broad bean mottle virus and cowpea chlorotic mottle virus.
The complete nucleotide sequences of the RNA-1 segments in broad bean mottle virus (BBMV) and cowpea chlorotic mottle virus (CCMV) were determined. BBMV RNA-1 consists of 3158 nucleotides and CCMV RNA-1 has 3171 nucleotides. Both BBMV and CCMV RNA-1 are capped at the 5' end but, unlike in other tricornaviruses, BBMV RNA-1 initiates with an A residue. Both BBMV and CCMV RNA-1 are monocistronic encoding for highly homologous 1a proteins of 966 and 958 amino acids, respectively. The highest homologies are clustered within two domains: the N-domain that aligns with the nsP1 Sindbis virus protein, a putative methyl transferase, and the C-domain which has a conserved nucleotide binding motif. Previous sequence comparisons suggest that the C-terminal domain may function as an NTP-dependent RNA helicase. In addition, we find that the C-domain has patterns similar to those of the reovirus and vaccinia virus guanylyl transferases. All this implies that 1a protein is a multifunctional polypeptide involved in both RNA capping and RNA polymerization processes.
Amino Acid Sequence↗
Specificity of recognition of mRNA 5' cap by human nuclear cap-binding complex.
The heterodimeric nuclear cap-binding complex (CBC) binds to the mono-methylated 5' cap of eukaryotic RNA polymerase II transcripts such as mRNA and U snRNA. The binding is important for nuclear maturation of mRNAs and possibly in the first round of translation and nonsense-mediated decay. It is also essential for nuclear export of U snRNAs in metazoans. We report characterization by fluorescence spectroscopy of the recognition of 5' capped RNA by human CBC. The association constants (K(as)) for 17 mono- and dinucleotide cap analogs as well as for the oligomer m7GpppA(m2') pU(m2')pA(m2') cover the range from 1.8 x 10(6) M(-1) to 2.3 x 10(8) M(-1). Higher affinity for CBC is observed for the dinucleotide compared with mononucleotide analogs, especially for those containing a purine nucleoside next to m7G. The mRNA tetramer associates with CBC as tightly as the dinucleotide analogs. Replacement of Tyr138 by alanine in the CBP20 subunit of CBC reduces the cap affinity except for the mononucleotide analogs, consistent with the crystallographic observation of the second base stacking on this residue. Our spectroscopic studies showed that contrary to the other known cap-binding proteins, the first two nucleotides of a capped-RNA are indispensable for its specific recognition by CBC. Differences in the cap binding of CBC compared with the eukaryotic translation initiation factor 4E (eIF4E) are analyzed and discussed regarding replacement of CBC by eIF4E.
Binding Sites↗
Sendai virus RNA-dependent RNA polymerase L protein catalyzes cap methylation of virus-specific mRNA.
The Sendai virus (SeV) RNA-dependent RNA polymerase complex, which consists of L and P proteins, participates in the synthesis of viral mRNAs that possess a methylated cap structure. To identify the SeV protein(s) involved in mRNA cap methylation, we developed an in vitro assay system to detect mRNA (guanine-7-)methyltransferase (G-7-MTase) activity. Viral ribonucleoprotein complexes and purified recombinant L protein but not P protein exhibited G-7-MTase activity. On the other hand, mRNA synthesis in a reconstituted transcription system using purified N-RNA (N protein-genomic RNA) complex as a template required both the L and P proteins. The enzymatic properties of SeV G-7-MTase were different from those of cellular G-7-MTase. In particular, unlike cellular G-7-MTase, the SeV enzyme preferentially methylated capped RNA containing the viral mRNA 5'-end sequences (GpppApGpG-). The C-terminal part (amino acid residues 1,756-2,228) of the L protein catalyzed cap methylation, whereas the N-terminal half (residues 1-1,120) containing putative RNA polymerase subdomains did not. This is to our knowledge the first direct biochemical evidence that supports the idea that mononegavirus L protein catalyzes cap methylation as well as RNA synthesis.
Baculoviridae↗
Absence of 5' terminal capping in encephalomyocarditis virus RNA.
The nature of the 5' terminus of encephalomyocarditis (EMC) virion RNA has been investigated. We have failed to detect any capped products or nucleoside polyphosphates arising upon complete digestion of the RNA with T1, T2, and pancreatic ribonucleases, and it would therefore appear that the 5' terminus of EMC virus RNA is not phosphorylated and not capped with m7G. EMC virions do contain, however, large amounts of all four 5'-monosubstituted nucleoside triphosphates (4.2M pppG; 16.4M pppA; 3.OM pppU and 2.5M pppC), of which at least a proportion (about 15-20%) appear to remain bound to fully denatured RNA in the presence of divalent cations.
Base Sequence↗
Light-induced switch in barley psbD-psbC promoter utilization: a novel mechanism regulating chloroplast gene expression.
The synthesis of reaction center protein D2 and mRNAs which encode this protein are differentially maintained at high levels in mature barley chloroplasts. To understand the differential maintenance of psbD mRNA abundance, we have studied the transcription and the RNAs produced from the psbD-psbC operon in plastids of light and dark-grown barley seedlings. Ten psbD-psbC RNAs synthesized in dark-grown barley share four different 5'-ends, two of which arise by transcription initiation, and one of which is generated by 5'-processing of longer psbD-psbC transcripts. Illumination of dark-grown barley causes the decline of these ten transcripts, and the accumulation of two different psbD-psbC RNAs. Capping assays, in vitro transcription and RNA processing experiments and treatment of plants with tagetitoxin (a selective inhibitor of chloroplast transcription), indicate that the light-induced transcripts arise by transcription initiation. Run-on transcription and RNA quantitation experiments provide evidence that both light-induced transcription and RNA stability play roles in the accumulation of the light-induced RNAs. These data document a novel mechanism for regulating plastid gene expression involving a light-induced switch in psbD-psbC promoter utilization.
Base Sequence↗
Characterization of the 5'-terminal cap structures of early simian virus 40 mRNA.
Early simian virus 40-specific mRNA was isolated from lytically infected and stably transformed cells and analyzed with respect to the 5'-terminal cap content. An analogous diversity of capped structures was found in both RNA preparations, indicating that 5'-terminal heterogeneity is not limited to late simian virus 40 RNA transcription but concerns a more general feature. Correlation of our data with previously published results of characterization of early mRNA's by reverse transcription allowed a tentative localization of the major 5' termini on the viral genome.
Animals↗
Effects of cap analogue or cap removal on the translation of rat brain mRNA in vitro.
The role of cap structures in the translation of brain mRNA was examined by measuring protein biosynthesis in vitro in wheat germ and reticulocyte systems programmed by mRNA that was either untreated or oxidized by periodate or from which 5'-terminal 7-methylguanosine (m7G) was removed by oxidation and beta-elimination. In another series of reactions, amino acid incorporation into polypeptides was measured in the absence and in the presence of varying concentrations of the cap analogue 7-methylguanosine 5'-triphosphate (pppm7G). The results indicated that any of the above treatments interfered with brain mRNA translation, the degree of inhibition depending on the translation system used, the concentration of mRNA, and the source of initiation factors. Homologous brain initiation factors were superior to reticulocyte factors in providing a partial relief from inhibition of translation caused by these treatments. It was also found that synthesis of the brain-specific protein S-100 was inhibited by beta-elimination of mRNA, by pppm7G, or by the presence of capped globin mRNA, indicating that the mRNA for this protein was probably capped.
Animals↗
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↗
Transcription of the 5'-terminal cap nucleotide by RNA-dependent DNA polymerase: possible involvement in retroviral reverse transcription.
The possible transcription of the 5'-terminal cap nucleotide of mRNA by RNA-dependent DNA polymerase was examined by a single-step assay, based on the generation of a hairpin structure during reverse transcription of globin mRNA. Using this approach, we demonstrated that the 5'-terminal cap nucleotide of mRNA is indeed transcribed by RNA-dependent DNA polymerase into a 3'-terminal residue of cDNA and were able to measure the extent of such transcription. The observed transcription of the cap nucleotide raises a number of questions that may be addressed using the relatively simple single-step assay employed in the present study. Cap nucleotide transcription by RNA-dependent DNA polymerase may have important implications for our understanding of the mechanism of action of reverse transcriptases. It may represent a selection mechanism for only partial transcription of the 5' repeat element of viral RNA genome, thus generating a RNA fragment that may play a role in priming of second (plus) DNA strand during retroviral reverse transcription. Moreover, the demonstrated ability of complementary nucleotides to form hydrogen bonds, even when in a parallel orientation, may have interesting and important consequences.
Animals↗
Mature mRNAs of Trypanosoma brucei possess a 5' cap acquired by discontinuous RNA synthesis.
Mature mRNAs of Trypanosoma brucei have a common 5' terminal sequence of 35 nucleotides. This is acquired by an unknown mechanism from the 5' end of a separately transcribed precursor RNA of about 140 nt called the mini-exon-derived RNA or medRNA. We have investigated the nature of the 5' ends of mature mRNAs and of the medRNA by chemical decapping and enzymic recapping. We infer that a 5' cap is present on both of these RNAs and conclude that the mini-exon-derived RNA donates its 5' cap along with the mini-exon sequence to the pre-mRNA. Using nuclear run-on experiments we show that medRNA synthesis is much more sensitive to alpha-amanitin than 5S RNA synthesis and only slightly less sensitive than tubulin gene transcription. This result, together with the presence of a cap at the 5' end of the medRNA indicates that the mini-exon is transcribed by an RNA polymerase II type enzyme. Our experiments also confirm the existence of a second minor medRNA of about 125 nt and show the presence of other small capped RNAs possibly analogous to the small nuclear RNAs of other organisms.
Amanitins↗
The 1.85 A structure of vaccinia protein VP39: a bifunctional enzyme that participates in the modification of both mRNA ends.
VP39 is a bifunctional vaccinia virus protein that acts as both an mRNA cap-specific RNA 2'-O-methyltransferase and a poly(A) polymerase processivity factor. Here, we report the 1.85 A crystal structure of a VP39 variant complexed with its AdoMet cofactor. VP39 comprises a single core domain with structural similarity to the catalytic domains of other methyltransferases. Surface features and mutagenesis data suggest two possible RNA-binding sites with novel underlying architecture, one of which forms a cleft spanning the region adjacent to the methyltransferase active site. This report provides a prototypic structure for an RNA methyltransferase, a protein that interacts with the mRNA 5' cap, and an intact poxvirus protein.
Binding Sites↗