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PubMed2003

Crucial role of CA cleavage sites in the cap-snatching mechanism for initiating viral mRNA synthesis.

In viral cap-snatching, the endonuclease intrinsic to the viral polymerase cleaves cellular capped RNAs to generate capped fragments that are primers for viral mRNA synthesis. Here we demonstrate that the influenza viral polymerase, which is assembled in human cells using recombinant proteins, effectively uses only CA-terminated capped fragments as primers for viral mRNA synthesis in vitro. Thus we provide the first in vitro system that mirrors the cap-snatching process occurring in vivo during virus infection. Further, we demonstrate that when a capped RNA substrate contains a CA cleavage site, the functions of virion RNA (vRNA) differ from those previously described: the 5' terminal sequence of vRNA alone is sufficient for endonuclease activation, and the 3' terminal sequence of vRNA functions solely as a template for mRNA synthesis. Consequently, we are able to identify the vRNA sequences that are required for each of these two separable functions. We present a new model for the influenza virus cap-snatching mechanism, which we postulate is a paradigm for the cap-snatching mechanisms of other segmented, negative-strand and ambisense RNA viruses.

Base Pairing↗

PubMed1989

Characterization of primary transcripts and identification of transcription initiation sites on the heavy and light strands of mouse mitochondrial DNA.

Total RNA from Ehrlich ascites mitochondria pretreated with RNase-free DNase was capped in vitro with [alpha-32P]GTP and guanylyl transferase. The cappable RNAs representing the primary transcripts show a heterogeneous size distribution with four major species of 46, 63, 94, and 152 nucleotides and four minor species of 19, 24, 104, and 790 nucleotides in size. Hybridization with the D-loop DNA probes shows that the 19-nucleotide-long capped RNA is coded by the H-strand of mitochondrial DNA while the rest are coded by the L-strand. S1 nuclease mapping and primer extension analyses suggest the occurrence of a transcription initiation of H-strand at about 19 nucleotides upstream from the start of the tRNA(Phe) gene. All of the L-strand cappable RNAs have a common 5' end mapping to nucleotide 16,183 +/- 5 of the genome. The 3' ends of four major cappable RNA species line up to the conserved sequence boxes, putative start sites of DH-DNA; and in fact about 2% of these cappable species are found to exist as DNA-linked RNA under steady-state conditions. The 3' end of the 790-nucleotide cappable RNA lies close to the start of the tRNA(Pro) gene, suggesting that it may be the true precursor of L-strand transcript endonucleolytically processed at the 3' end. The level of L-strand-coded cappable RNAs varies markedly under different growth conditions. Treatment with cycloheximide results in a reduction while chloramphenicol caused over 3-fold induction, suggesting that these "primer" RNAs may have an additional regulatory function.

Animals↗

PubMed1982

Purification and characterization of the messenger ribonucleic acid capping enzyme GTP:RNA guanylyltransferase from wheat germ.

A GTP:RNA guanylyltransferase or capping enzyme has been purified approximately 2000-fold from wheat germ. The enzyme catalyzes the transfer of the GMP residue from GTP to the 5' end of RNA or synthetic polyribonucleotides. Diphosphate-ended polymers were capped more efficiently than molecules with triphosphate ends, and molecules with monophosphate ends were not capped at all. There appears to be little specificity since RNAs with purine or pyrimidine ends served as acceptors. Other features of the wheat germ RNA guanylyltransferase include relatively low Km values for GTP (2.7 microM) and ppA (pA)n (14.2 nM), a divalent cation requirement satisfied by low (0.5 mM) concentrations of MnCl2 or higher (5 mM) concentrations of MgCl2, and a pH optimum around neutrality.

Kinetics↗

PubMed1995

Synthesis of an infectious full-length cDNA clone of rice yellow mottle virus and mutagenesis of the coat protein.

A full-length cDNA clone of rice yellow mottle sobemovirus (RYMV) was synthesized and placed adjacent to a bacteriophage T7 RNA polymerase promoter sequence. Capped-RNA transcripts produced in vitro were infectious when mechanically inoculated onto rice plants (Oryza sativa L). Individual full-length clones varied in their degree of infectivity but all were less infectious than native viral RNA. A representative clone, designated RYMV-FL5, caused a disease phenotype identical to that produced by viral RNA except that symptoms were somewhat slower to appear than those induced by viral RNA. The infectivity of RYMV-FL5 was verified by ELISA, Western blot analysis, Northern blot hybridization, RT-PCR, and Southern blot hybridization. Frameshift and deletion mutations introduced into the coat protein cistron demonstrated that the coat protein was dispensable for RNA replication in rice protoplasts. However, the coat protein was required for full infectivity in rice plants, presumably by playing a role in phloem-mediated long-distance movement and possibly in cell-to-cell movement.

Base Sequence↗

PubMed2001

Influenza virus endoribonuclease.

The influenza virus polymerase complex contains two associated enzymatic activities, an endoribonuclease and a RNA-dependent RNA polymerase activity. Both activities have so far been observed only with the complete polymerase complex consisting of three subunits, PB1, PB2, and PA. This chapter describes a robust and optimized procedure for the purification of active influenza virus polymerase in complex with genomic RNA and the single-stranded RNA-binding protein nucleoprotein from influenza virus particles. It also explains the synthesis of capped RNA molecules as substrates of the influenza virus endonuclease. The enzymatic properties of influenza virus-derived endoribonuclease activity have been characterized with a model RNA substrate of 20-nucleotide length, termed G20 RNA. The rate of RNA cleavage under steady state conditions appears to be limited by product dissociation. Therefore conditions have been optimized to study the chemical step of RNA cleavage under single turnover conditions. The enzyme requires divalent metal ions for activity and can use Mn(II), Co(II), and Fe(II) efficiently at pH 7, Mg(II) with intermediate efficiency, and Ni(II) and Zn(II) with lower efficiency. The reaction progress curves show slow binding of Zn(II) and Ni(II) to the protein, suggesting a conformational change of the active site as a prerequisite for endonuclease activity in the presence of these two metal ions. Low concentrations of the detergent DOC inhibit the activity and also disrupt the trimeric polymerase complex, whereas other detergents do not have a significant effect on the activity.

Base Sequence↗

PubMed1996

Vaccinia virus mRNA (guanine-7-)methyltransferase: mutational effects on cap methylation and AdoHcy-dependent photo-cross-linking of the cap to the methyl acceptor site.

The (guanine-7-)methyltransferase domain of the vaccinia virus mRNA capping enzyme is composed of the C-terminal portion of the D1 subunit, D1(498-844), heterodimerized with the D12 protein. In order to identify protein structural elements involved in cap methylation, we introduced eight alanine substitution mutations within two sequence motifs of D1(498-844)-(594)VLAIDFGNG(602) and (681)IHYSF(685)--that are conserved in the cap methyltransferase from yeast. The D1(498-844)-Ala proteins were coexpressed in bacteria with the D12 subunit, and the recombinant D1(498-844)/D12 heterodimers were purified. Alanine substitutions at five positions--Asp-598, Gly-602, Ile-681, Ser-684, and Phe-685--had little or no effect on methyltransferase activity. Mutations at three conserved residues were deleterious. Alanine substitution at Gly-600 reduced the specific activity to 4% of that of the wild-type protein. Substitutions at His-682 and Tyr-683 reduced activity to 4% and 0.05%, respectively. By further mutating Tyr-683 to Phe and Ser, we established that the aromatic group was essential for cap methylation, whereas the hydroxyl moiety was dispensable. Specific binding of the methyltransferase to the RNA cap was demonstrated by UV cross-linking to [32P]GMP-labeled capped poly(A). Label transfer occurred exclusively to the D1(498-844) subunit and was competed by the cap analogs GpppA and m7GpppA. Cap-specific cross-linking to m7GpppA(pA)n was stimulated by AdoHcy, whereas cross-linking to GpppA(pA)n was unaffected by AdoHcy, but stimulated by AdoMet. We suggest that occupancy of the methyl donor site either enhances the affinity for the cap guanosine or alters the protein interface so that a photoreactive moiety is brought closer to the cap structure. The catalytically defective H682A, Y683A, and Y683S mutant methyltransferases were unable to cross-link to the cap in the presence of AdoHcy. The catalytically defective G600A mutant did cross-link to the cap in the presence of AdoHcy, suggesting that this mutation affects the chemical step of transmethylation.

Alanine↗

PubMed1997

The role of the cap structure in RNA processing and nuclear export.

The cap structure that is characteristic of all polymerase-II-transcribed RNAs has been shown to play an important role in many aspects of RNA metabolism including RNA processing, RNA nuclear transport, and translation initiation. The effects of the cap structure on these different processes is mediated by proteins that recognise and bind to it, and are therefore generically called cap-binding proteins. For example, the cap-binding protein eIF4E, in a complex with other proteins, mediates the effect of the cap on the initiation of translation. EIF-4E is predominantly localised in the cytoplasm. In the last five years, it has been demonstrated that a second cap-binding protein complex, which is mainly localised in the nucleus, mediates the stimulatory effects of the cap in nuclear processes such as pre-mRNA splicing, RNA 3'-end formation, and RNA nuclear export. The purpose of this review is to summarise our current knowledge on the role of the cap structure and of the cap-binding protein complex in nuclear RNA metabolism and present evidence that at least some processes may be coupled in vivo.

Animals↗

PubMed2005

Processing the message: structural insights into capping and decapping mRNA.

The 5' m7GpppN cap plays an essential role in the life cycle of eukaryotic mRNA and is required for efficient pre-mRNA splicing, export, stability and translation. Nascent pre-mRNA is capped through a series of three enzymatic steps that result in a 5' N7-methyl guanosine linked by an inverted 5'-5' triphosphate bridge to the first nucleotide of the transcript. Early structural studies revealed the mechanisms employed in the first two steps of capping, and more recent structural studies have completed the suite of capping activities and shed light on the mechanisms that target the capping apparatus to the phosphorylated C-terminal domain of RNA polymerase II. RNA decay pathways also target the RNA cap structure. After deadenylation of polyadenylated mRNA, enzymes of the 5'-3' decay pathway hydrolyze the mRNA cap to expose the 5' RNA end to 5'-3' exoribonuclease activities. In the 3'-5' decay pathway, exosome-mediated degradation of RNA occurs from the 3' end after deadenylation, ultimately generating a cap structure that is hydrolyzed by enzymes of this pathway. Recent structural studies have illuminated the mechanisms employed for decapping mRNA in both 5'-3' and 3'-5' decay pathways.

Acid Anhydride Hydrolases↗

PubMed1981

Transcriptional initiation and 5' termini of yeast mitochondrial RNA.

We have used vaccinia virus guanylyltransferase to label polyphosphate-terminated yeast mitochondrial RNAs in vitro with [alpha-32P]GTP. Hybridization of RNA labeled in vitro indicates the presence of multiple transcriptional initiation sites in both grande and petite mitochondrial genomes. Agarose/urea gel electrophoresis of capped RNA suggests the existence of a precursor to the small (14 S) rRNA. In contrast, direct examination of the large (21 S) rRNA by partial ribonuclease T1 digestion reveals a complete lack of processing of the 5' end of the primary transcript of this RNA.

DNA Restriction Enzymes↗

PubMed1990

Monomethylated cap structures facilitate RNA export from the nucleus.

RNA export from the nucleus has been analyzed in Xenopus oocytes. U1 snRNAs made by RNA polymerase II were exported into the cytoplasm, while U1 snRNAs synthesized by RNA polymerase III, and therefore with a different cap structure, remained in the nucleus. Export of the polymerase II-transcribed RNAs was inhibited by the cap analog m7GpppG. Spliced mRNAs carrying monomethylguanosine cap structures were rapidly exported, while hypermethylated cap structures delayed mRNA export. The export of a mutant precursor mRNA unable to form detectable splicing complexes was also significantly delayed by incorporation of a hypermethylated cap structure. The results suggest that the m7GpppN cap structure is likely to be a signal for RNA export from the nucleus.

Animals↗

PubMed1987

In vitro synthesis of influenza viral RNA: characterization of an isolated nuclear system that supports transcription of influenza viral RNA.

An in vitro system for the synthesis of influenza viral RNA was developed using isolated nuclei prepared from influenza virus-infected HeLa cells. In this system, two species of positive-sense RNA, i.e., mRNA and cRNA (complementary RNA to vRNA), were found to be synthesized when analyzed by RNA-RNA hybridization using a minus-strand RNA probe and high resolution gel electrophoresis. The in vitro RNA synthesis required Mg2+, GTP, CTP, UTP, a high concentration of ATP, and an ATP regenerating system. Neither actinomycin D nor alpha-amanitin, potent inhibitors for cellular DNA-dependent RNA polymerases, inhibited the RNA synthesis. Addition of ApG or capped RNA, well-known primers for virion-associated RNA polymerase, markedly enhanced the extent of RNA synthesis. The maximum activity was observed with nuclei isolated from cells at 5 h after infection. This system is useful for the purification and characterization of factors involved in the transcription of these two species positive-sense RNA.

Adenosine Triphosphate↗

PubMed1990

Capping of tobacco mosaic virus RNA. Analysis of viral-coded guanylyltransferase-like activity.

The 5' end of tobacco mosaic virus (TMV) genomic RNA is capped with 7-methylguanosine. A virus-coded polypeptide with guanylyltransferase activity has been investigated. This enzyme is responsible for forming the 5'----5' linkage of guanosine 5'-monophosphate to the 5'-diphosphate of an acceptor RNA, thereby forming the cap. A critical step in the mechanism for cap formation in the eukaryotic nucleus is for guanylyltransferase to bind covalently to guanosine 5'-monophosphate with the hydrolysis of pyrophosphate when guanosine 5'-triphosphate is the substrate. The TMV 126-kilodalton protein, which is most probably a component of the TMV replicase, was found to have this activity. The mechanism of this reaction has been characterized biochemically.

Binding, Competitive↗

PubMed1997

X-ray studies of the messenger RNA 5' cap-binding protein (eIF4E) bound to 7-methyl-GDP.

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.

Crystallography, X-Ray↗

PubMed1999

Structure and mechanism of yeast RNA triphosphatase: an essential component of the mRNA capping apparatus.

RNA triphosphatase is an essential mRNA processing enzyme that catalyzes the first step in cap formation. The 2.05 A crystal structure of yeast RNA triphosphatase Cet1p reveals a novel active site fold whereby an eight-stranded beta barrel forms a topologically closed triphosphate tunnel. Interactions of a sulfate in the center of the tunnel with a divalent cation and basic amino acids projecting into the tunnel suggest a catalytic mechanism that is supported by mutational data. Discrete surface domains mediate Cet1p homodimerization and Cet1p binding to the guanylyltransferase component of the capping apparatus. The structure and mechanism of fungal RNA triphosphatases are completely different from those of mammalian mRNA capping enzymes. Hence, RNA triphosphatase presents an ideal target for structure-based antifungal drug discovery.

Acid Anhydride Hydrolases↗

PubMed1997

Isolation of novel ribozymes that ligate AMP-activated RNA substrates.

BACKGROUND: The protein enzymes RNA ligase and DNA ligase catalyze the ligation of nucleic acids via an adenosine-5'-5'-pyrophosphate 'capped' RNA or DNA intermediate. The activation of nucleic acid substrates by adenosine 5'-monophosphate (AMP) may be a vestige of 'RNA world' catalysis. AMP-activated ligation seems ideally suited for catalysis by ribozymes (RNA enzymes), because an RNA motif capable of tightly and specifically binding AMP has previously been isolated. RESULTS: We used in vitro selection and directed evolution to explore the ability of ribozymes to catalyze the template-directed ligation of AMP-activated RNAs. We subjected a pool of 10(15) RNA molecules, each consisting of long random sequences flanking a mutagenized adenosine triphosphate (ATP) aptamer, to ten rounds of in vitro selection, including three rounds involving mutagenic polymerase chain reaction. Selection was for the ligation of an oligonucleotide to the 5'-capped active pool RNA species. Many different ligase ribozymes were isolated; these ribozymes had rates of reaction up to 0.4 ligations per hour, corresponding to rate accelerations of approximately 5 x10(5) over the templated, but otherwise uncatalyzed, background reaction rate. Three characterized ribozymes catalyzed the formation of 3'-5'-phosphodiester bonds and were highly specific for activation by AMP at the ligation site. CONCLUSIONS: The existence of a new class of ligase ribozymes is consistent with the hypothesis that the unusual mechanism of the biological ligases resulted from a conservation of mechanism during an evolutionary replacement of a primordial ribozyme ligase by a more modern protein enzyme. The newly isolated ligase ribozymes may also provide a starting point for the isolation of ribozymes that catalyze the polymerization of AMP-activated oligonucleotides or mononucleotides, which might have been the prebiotic analogs of nucleoside triphosphates.

Adenosine Monophosphate↗

PubMed1985

Sindbis virus mutants able to replicate in methionine-deprived Aedes albopictus cells.

Previous work from this laboratory has shown that the replication of Sindbis virus (SV) in Aedes albopictus cells is sensitive to methionine withdrawal. This sensitivity is thought to reflect a diminished concentration of S-adenosylmethionine (Ado Met) resulting from methionine starvation. Serial passage of SV on Ae. albopictus cells maintained in low concentrations of methionine gave rise to a population of mutants whose replication in mosquito cells was resistant to methionine starvation. In vertebrate cells, these mutants were also resistant to inhibition by cycloleucine. We favor the hypothesis that the adaptation to low methionine reflects the accumulation of mutations resulting in a viral RNA "cap" methyltransferase with an increased affinity for Ado Met.

Aedes↗

PubMed2025

Transcriptome-Wide Analysis of the 5' Cap Status of RNA Using 5' Monophosphate-Dependent Exonuclease Digestion and RNA Sequencing.

Eukaryotic mRNAs carry an N7-methylguanosine (m7G) cap structure at their 5' extremity, which protects them from the degradation by 5'-3' exoribonucleases and plays a pivotal role in mRNA metabolism, promoting splicing, nuclear export, and translation. Decapping, the enzymatic process that removes this structure, is a key event during cytoplasmic mRNA 5'-3' decay, leading to the degradation of the transcript body by Xrn1. In this chapter, we describe a procedure to assess the cap status of RNA at the transcriptome level. It is based on a treatment of total RNA extracts with a 5' monophosphate-dependent exonuclease, which like Xrn1 specifically degrades decapped RNAs harboring 5' monophosphate extremities, but not RNAs with intact m7G cap. The digested RNAs are then analyzed by RNA sequencing.

Exoribonucleases↗

PubMed2003

Evaluation of the RNA determinants for bacterial and yeast RNase III binding and cleavage.

Bacterial double-stranded RNA-specific RNase III recognizes the A-form of an RNA helix with little sequence specificity. In contrast, baker yeast RNase III (Rnt1p) selectively recognizes NGNN tetraloops even when they are attached to a B-form DNA helix. To comprehend the general mechanism of RNase III substrate recognition, we mapped the Rnt1p binding signal and directly compared its substrate specificity to that of both Escherichia coli RNase III and fission yeast RNase III (PacI). Rnt1p bound but did not cleave long RNA duplexes without NGNN tetraloops, whereas RNase III indiscriminately cleaved all RNA duplexes. PacI cleaved RNA duplexes with some preferences for NGNN-capped RNA stems under physiological conditions. Hydroxyl radical footprints indicate that Rnt1p specifically interacts with the NGNN tetraloop and its surrounding nucleotides. In contrast, Rnt1p interaction with GAAA-capped hairpins was weak and largely unspecific. Certain duality of substrate recognition was exhibited by PacI but not by bacterial RNase III. E. coli RNase III recognized RNA duplexes longer than 11 bp with little specificity, and no specific features were required for cleavage. On the other hand, PacI cleaved long, but not short, RNA duplexes with little sequence specificity. PacI cleavage of RNA stems shorter than 27 bp was dependent on the presence of an UU-UC internal loop two nucleotides upstream of the cleavage site. These observations suggest that yeast RNase IIIs have two recognition mechanisms, one that uses specific structural features and another that recognizes general features of the A-form RNA helix.

Base Sequence↗