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PubMed1991

A nuclear micrococcal-sensitive, ATP-dependent exoribonuclease degrades uncapped but not capped RNA substrates.

We have developed an assay for an exoribonuclease present in HeLa cell nuclear extracts that degrades capped but not uncapped RNA substrates, and used it to partially purify and characterize such an activity. Capped and uncapped transcripts of different sizes (37-317 nt) were incubated with fractionated nuclear extracts, and in all cases the capped RNAs were stable while their uncapped counterparts were completely degraded. No changes in activity were detected when cap analogs were included in reaction mixtures, suggesting that the stability of capped RNAs was not due to a cap binding protein. The exoribonuclease was shown to be specific for RNA, and to function processively with either substrates containing 5'-hydroxyl or 5'-phosphorylated ends. The products were predominantly 5'-mononucleotides, and no detectable intermediates were observed at any reaction time points. Sedimentation analysis suggests that the native size of the nuclease is 7.4S or approximately 150 kDa. Interestingly, a nucleoside triphosphate was found to be necessary for specific and complete degradation of the uncapped RNAs. Finally, micrococcal nuclease (MN) pretreatment of the partially purified enzyme inhibited its activity. As several controls indicated that this was not due to non-specific effects of MN, this finding suggests that the exoribonuclease contains an essential RNA component.

Adenosine Triphosphate↗
PubMed2002

What messenger RNA capping tells us about eukaryotic evolution.

The 5' cap is a unique feature of eukaryotic cellular and viral messenger RNA that is absent from the bacterial and archaeal domains of life. The cap is formed by three enzymatic reactions at the 5' terminus of nascent mRNAs. Although the capping pathway is conserved in all eukaryotes, the structure and genetic organization of the component enzymes vary between species. These differences provide insights into the evolution of eukaryotes and eukaryotic viruses.

Acid Anhydride Hydrolases↗
PubMed1982

Simple, efficient in vitro synthesis of capped RNA useful for direct expression of cloned eukaryotic genes.

A simple and efficient method for direct in vitro synthesis of capped transcripts of cloned eukaryotic genes is described. As an example capped transcripts were made from a plasmid containing the human fibroblast interferon gene cloned under the control of a prokaryotic promoter. These transcripts were translated in vivo in Xenopus laevis oocytes and in vitro in reticulocyte and in wheat germ cell-free protein synthesizing systems.

Animals↗
PubMed1996

Domain analysis of human U5 RNA. Cap trimethylation, protein binding, and spliceosome assembly.

We have analyzed the sequence requirements of the human U5 RNA during small nuclear ribonucleoprotein (snRNP) and spliceosome assembly. A collection of mutant derivatives of the human U5 RNA gene was constructed in a U1 expression vector and transiently transfected in mammalian cells. Using immunoprecipitation and affinity selection assays, the cap trimethylation, the binding of Sm proteins and of the U5 snRNP-specific protein p220, as well as the assembly of the U4/U5/U6 triple snRNP and of spliceosomes were determined. By mutational analysis we were able to assign distinct functions to several structural elements of the human U5 RNA. Efficient binding of the Sm proteins requires the 3' stem-loop. Both the Sm protein-binding site and the 3' stem-loop are necessary for the formation of the trimethyl guanosine cap, consistent with Sm protein binding being a prerequisite for cap trimethylation. Specific elements of the U5 RNA 5' stem-loop contribute to efficient p220 association, in particular stem Ib. Interestingly, the highly conserved loop I appears to be a multifunctional element; in addition to its function in splice-site selection the 5' loop is involved in binding of p220 and in the assembly of the U4/U5/U6 triple snRNP. In sum, this mutational analysis has identified four functional domains of the human U5 RNA.

Animals↗
PubMed2003

Interaction between the small-nuclear-RNA cap hypermethylase and the spinal muscular atrophy protein, survival of motor neuron.

The biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) requires the cytoplasmic assembly of the Sm-core complex, followed by the hypermethylation of the small nuclear RNA (snRNA) 5' cap. Both the Sm-core complex and the snRNA trimethylguanosine cap are required for the efficient nuclear import of snRNPs. Here, we show that trimethylguanosine synthase 1 (TGS1), the human homologue of the yeast snRNA cap hypermethylase, interacts directly with the survival of motor neuron (SMN) protein. Both proteins are similarly distributed, localizing in the cytoplasm and in nuclear Cajal bodies. The interaction between TGS1 and SMN is disrupted by a mutation in SMN that mimics the predominant isoform of the protein that is expressed in patients with the neurodegenerative disease, spinal muscular atrophy. These data indicate that, in addition to its function in cytoplasmic Sm-core assembly, the SMN protein also functions in the recruitment of the snRNA cap hypermethylase.

Autoantigens↗
PubMed1999

Putative RNA capping activities encoded by brome mosaic virus: methylation and covalent binding of guanylate by replicase protein 1a.

Brome mosaic virus (BMV) RNA replication is directed by two virus-encoded proteins, 1a and 2a. The amino-terminal half of 1a is a distant homolog of alphavirus nonstructural protein nsP1, which has been implicated in capping viral RNAs. In this study, we examined the enzymatic activities of BMV 1a expressed in yeast, where the protein is fully functional in RNA replication. 1a methylated GTP, dGTP, and the cap analogs GpppG and GpppA, using S-adenosylmethionine (AdoMet) as the methyl donor. Product analysis by nuclear magnetic resonance spectroscopy showed that 1a methylation was specific for guanine position 7. Additionally, 1a interacted with GTP to form a covalent 1a-m(7)GMP complex. This reaction was specific for GTP, required AdoMet, and was accompanied by transfer of (3)H-methyl from AdoMet to the covalent 1a-guanylate complex. The covalent complex could be immunoprecipitated by 1a antibodies. The 1a-m(7)GMP complex was inhibited in catalyzing further methyltransferase reactions. Mutation of conserved amino acids in the N-terminal half of 1a reduced both methyltransferase and covalent complex formation activities to very low or undetectable levels. Covalent 1a-guanylate complex formation took place in similar, AdoMet-dependent fashion in extracts of BMV-infected barley protoplasts. These results show that BMV 1a has activities similar to those of alphavirus nsP1, demonstrating conservation of these putative capping functions across a wide span of sequence divergence within the alphavirus-like superfamily. Conservation of this unusual combination of functions also supports the inference that the superfamily caps viral RNAs by an unusual pathway proceeding via a m(7)GMP intermediate.

Amino Acid Sequence↗
PubMed1977

Studies on the structure and possible function of the RNA "cap" in developing sea urchins.

Paracentrotus lividus embryos at the hatching blastula stage very quickly incorporate radioactivity from labeled nucleosides (except uridine) or 32P- or methyl-labeled methionine into a portion of the RNA that has been identified as a "cap". The most probable sequence of this cap is m7G (5') ppp (5')mAmpCp. A very active "capping" and methylation of the "cap" of preexisting RNA molecules was shown to occur at the blastula stage.

Animals↗
PubMed1985

Chemical synthesis of capped RNA fragments and their ability to complex with eukaryotic ribosomes.

In other to examine the binding ability of the 5'-terminal part of eukaryotic mRNA to 80S ribosome, several kinds of oligoribonucleotides, pA-U-G, m7G5'pppA-U-G, m7G5'pppG-U, m7G5'pppA-U-G-A-C-C, were synthesized chemically. The binding experiments of oligonucleotides to 80S ribosome showed that the capped structure as well as AUG are essential for ribosome binding, and the efficiency is enhanced by the 5'-leader sequence if it would include complementary sequence to the 3'-terminal part of 18S rRNA.

Base Sequence↗
PubMed1996

Expression and characterization of an RNA capping enzyme encoded by Chlorella virus PBCV-1.

We report that the A103R protein of Chlorella virus PBCV-1 is an mRNA capping enzyme that catalyzes the transfer of GMP from GTP to the 5' diphosphate end of RNA. This is a two-step reaction in which the enzyme first condenses with GTP to form a covalent enzyme-GMP intermediate and then transfers the GMP to an RNA acceptor to form a GpppN cap. Purified recombinant Al03R is a 38-kDa monomer that lacks RNA (guanine-7-) methyltransferase activity. With respect to its size, amino acid sequence, and biochemical properties, A103R is more closely related to the yeast RNA guanylyltransferases than it is to the multifunctional capping enzymes coded for by other large DNA viruses--the poxviruses and African swine fever virus. We surmise that in order to cap its transcripts, PBCV-l must either encode additional 5' processing activities or else rely on the host alga to provide these functions.

Chlorella↗
PubMed2000

A novel mechanism for inhibition of translation by pokeweed antiviral protein: depurination of the capped RNA template.

Pokeweed antiviral protein (PAP) is known to inactivate ribosomes by removal of a specific adenine from the sarcin/ricin (S/R) loop of the large rRNA, thereby inhibiting translation. We demonstrate here that in addition to the previously identified adenine (A4324), PAP removes another adenine (A4321) and a guanine (G4323) from the eukaryotic large rRNA. Recent results indicate that the antiviral activity of PAP may not be due to depurination of host ribosomes. Using PAP mutants that do not depurinate either tobacco or reticulocyte lysate rRNA, we show that PAP inhibits translation of brome mosaic virus (BMV) and potato virus X (PVX) RNAs without depurinating ribosomes. Furthermore, translation of only capped, but not uncapped, luciferase transcripts is inhibited by PAP, providing evidence that PAP and PAP mutants are able to distinguish between capped and uncapped transcripts. Translation inhibition of BMV RNAs is overcome by treatment with PAP in the presence of increasing concentrations of the cap analog m7GpppG, but not GpppG or GTP, indicating that PAP recognizes the cap structure. Incubation of BMV RNAs or the capped luciferase transcripts with PAP results in depurination of either RNA. In contrast, uncapped luciferase transcripts are not depurinated after incubation with identical concentrations of PAP. These results demonstrate for the first time that PAP can inhibit translation by a mechanism other than ribosome depurination, by recognizing the cap structure and specifically depurinating the capped RNAs.

Animals↗
PubMed1991

Direct introduction and transient expression of capped and non-capped RNA in Saccharomyces cerevisiae.

We report the introduction of functional RNA molecules into yeast spheroplasts. Plasmids containing the firefly luciferase coding region were transcribed to yield RNAs suitable for introduction into yeast cells and direct assay of their translation products. The 5' noncoding regions of the RNAs were derived either from the 5' noncoding regions of firefly luciferase, poliovirus, or yeast virus-like-particle (VLP) L-A or M1 RNAs. Capped and non-capped mRNAs were made by T7 RNA polymerase-directed transcription and introduced into yeast spheroplasts. The peak time of luciferase transient expression from introduced RNAs was 2-4 h after their introduction. In contrast, transient expression of luciferase from a non-replicative, luciferase-encoding plasmid introduced into the cells was maximal at 16 h. For capped mRNAs, luciferase activity increased linearly with transcript amount for both yeast and human (HeLa) cells. Although non-capped luciferase mRNAs were expressed more efficiently following introduction into yeast than into HeLa cells, the 5' noncoding sequences from yeast double-stranded (ds)RNA VLP RNAs conferred no greater apparent cap-independence than non-VLP RNA sequences in this transient expression assay. The RNA transient expression system will allow the study of translation of capped and non-capped RNAs in yeast cells and of the replicative cycle of yeast virus-like RNA genomes.

Base Sequence↗
PubMed2003

The packaging signal of simian immunodeficiency virus is upstream of the major splice donor at a distance from the RNA cap site similar to that of human immunodeficiency virus types 1 and 2.

Deletion mutation of the RNA 5' leader sequence of simian immunodeficiency virus (SIV) was used to localize the virus packaging signal. Deletion of sequences upstream of the major splice donor (SD) site produced a phenotype most consistent with a packaging defect when analysed by both RNase protection assay and RT-PCR. Sequences downstream of the SD were deleted and produced varying effects but did not affect packaging: a large downstream deletion had little effect on function, whereas a nested deletion produced a profound replication defect characterized by reduced protein production. Secondary structure analysis provided a potential explanation for this. The major packaging signal of SIV appears to be upstream of the SD in a region similar to that of human immunodeficiency virus type 2 (HIV-2) but unlike that of HIV-1; however, the packaging signal of all three viruses are at a similar distance from their respective cap sites. This conserved positioning suggests that it is more important in the virus life cycle than the position of the signal relative to the SD.

5' Untranslated Regions↗
PubMed1994

Covalent catalysis in nucleotidyl transfer reactions: essential motifs in Saccharomyces cerevisiae RNA capping enzyme are conserved in Schizosaccharomyces pombe and viral capping enzymes and among polynucleotide ligases.

Formation of the 5' cap structure of eukaryotic mRNAs occurs via transfer of GMP from GTP to the 5' terminus of the primary transcript. RNA guanylyltransferase, the enzyme that catalyzes this reaction, has been isolated from many viral and cellular sources. Though differing in molecular weight and subunit structure, the various guanylyltransferases employ a common catalytic mechanism involving a covalent enzyme-(Lys-GMP) intermediate. Saccharomyces cerevisiae CEG1 is the sole example of a cellular capping enzyme gene. In this report, we describe the identification and characterization of the PCE1 gene encoding the capping enzyme from Schizosaccharomyces pombe. PCE1 was isolated from a cDNA library by functional complementation in Sa. cerevisiae. Induced expression of PCE1 in bacteria and in yeast confirmed that the 47-kDa Sc. pombe protein was enzymatically active. The amino acid sequence of PCE1 is 38% identical (152 of 402 residues) to the 52-kDa capping enzyme from Sa. cerevisiae. Comparison of the two cellular capping enzymes with guanylyltransferases encoded by DNA viruses revealed local sequence similarity at the enzyme's active site and at four additional collinear motifs. Mutational analysis of yeast CEG1 demonstrated that four of the five conserved motifs are essential for capping enzyme function in vivo. Remarkably, the same motifs are conserved in the polynucleotide ligase family of enzymes that employ an enzyme-(Lys-AMP) intermediate. These findings illuminate a shared structural basis for covalent catalysis in nucleotidyl transfer and suggest a common evolutionary origin for capping enzymes and ligases.

Amino Acid Sequence↗
PubMed1996

The effects of palmitoylation on membrane association of Semliki forest virus RNA capping enzyme.

The nonstructural protein Nsp1 of Semliki Forest virus has guanine-7-methyltransferase and guanylyltransferase-like activities, required in the capping of viral mRNAs. It is palmitoylated and tightly associated with the cytoplasmic surface of the plasma membrane, endosomes, and lysosomes. To localize the acylation site(s) and the putative membrane-targeting domain, a number of deletions were made in the nsp1 gene. Most deletions resulted in the expression of nonpalmitoylated, enzymatically inactive, cytoplasmic protein. Palmitate could be released from Nsp1 with neutral hydroxylamine, indicating a thioester linkage to a cysteine residue. Therefore we mutated the conserved cysteine residues of Nsp1 to alanine. Triple mutation of Cys418, Cys419, and Cys420 resulted in nonpalmitoylated Nsp1, which was enzymatically active and still associated with membranes. However, it could be released from the membranes with 1 M NaCl, whereas 50 mM sodium carbonate (pH 12) was required to release wild type Nsp1, suggesting a conversion from an integral to a peripheral membrane protein. Indirect confocal immunofluorescence microscopy showed that the nonpalmitoylated Nsp1 colocalized with the plasma membrane marker, concanavalin A. However, it was not detected in filopodia, which were heavily stained in cells expressing wild type Nsp1. These results indicate that the acylation of Nsp1 was not needed for its targeting to the plasma membrane, but it was necessary for the migration to the filopodial extensions of the plasma membrane.

Acylation↗
PubMed2003

Autographa californica nucleopolyhedrovirus orf69 encodes an RNA cap (nucleoside-2'-O)-methyltransferase.

The AcNPV orf69 gene encodes a protein that contains an S-adenosylmethionine (AdoMet)-dependent methyltransferase signature motif. More significantly, ORF69 shows high conservation at residues diagnostic for (nucleoside 2'-O)-methyltransferase activity. To analyze the function of this protein, which was renamed MTase1, it was overexpressed in Escherichia coli and purified to homogeneity. Photo cross-linking experiments showed that MTase1 bound AdoMet, and functional assays demonstrated cap 0-dependent methyltransferase activity. In vivo expression assays in insect cells showed that MTase1 was synthesized during the late phase of infection and that its expression was dependent on viral DNA replication. Primer extension analysis identified a late promoter motif, ATAAG, at the transcription start site. A mutant virus was constructed by inserting the lacZ gene into the coding region of mtase1. Immunoblot analysis confirmed that MTase1 was not synthesized in these cells, and single-step growth curves revealed that the rate of virus replication in tissue culture was not affected by the absence of MTase1.

Amino Acid Sequence↗
PubMed1999

Two separate sequences of PB2 subunit constitute the RNA cap-binding site of influenza virus RNA polymerase.

BACKGROUND: Influenza virus RNA polymerase with the subunit composition of PB1-PB2-PA is a unique multifunctional enzyme with the activities of both synthesis and cleavage of RNA, and is involved in both transcription and replication of the RNA genome. Transcription is initiated by using capped RNA fragments, which are generated after cleavage of host cell mRNA by the RNA polymerase-associated capped RNA endonuclease. To identify the RNA cap 1-binding site on the RNA polymerase, viral ribonucleoprotein (RNP) cores were subjected to UV-crosslinking with RNA which was labelled with 32P only at the cap-1 structure. RESULTS: After SDS-PAGE of UV-crosslinked cores, 32P was found to be associated only with the PB2 subunit (759 amino acid residues). The labelled PB2 was subjected, together with PB2 expressed in E. coli, to limited digestion with V8 protease. Analysis of the amino terminal sequences of some isolated fragments with the crosslinked cap-1 indicated that two separate sequences within the PB2 were involved in RNA cap-1 binding, one (N-site) at the N-terminal proximal region approximately between amino acid residues 242-282 downstream from the PB1 subunit-binding site and the other (C-site) between residues 538-577 including the cap-binding motifs. Two lines of evidence support the prediction of the involvement of two separate PB2 sequences on the RNA cap-binding: (i) cross-linking of the capped RNA on to expressed and isolated PB2 fragments, each containing either the N-site or the C-site; and (ii) competition of capped RNA-binding to PB2 by both of the N- and C-terminal PB2 fragments. Taking together, we propose that two separate sequences within PB2 constitute the capped RNA-binding site of the RNA polymerase. CONCLUSION: Two separate sequences, one N-(242-282) and the other C-terminal (538-577) proximal segments of PB2 subunit, constitute the RNA cap-binding site of the influenza virus RNA polymerase.

Carrier Proteins↗
PubMed1990

Capping of mammalian U6 small nuclear RNA in vitro is directed by a conserved stem-loop and AUAUAC sequence: conversion of a noncapped RNA into a capped RNA.

The cap structure of U6 small nuclear RNA (snRNA) is gamma-monomethyl phosphate and is distinct from other known RNA cap structures (R. Singh and R. Reddy, Proc. Natl. Acad. Sci. USA 86:8280-8283, 1989). Here we show that the information for capping the U6 snRNA in vitro is within the initial 25 nucleotides of the U6 RNA. The capping determinant in mammalian U6 snRNA is a bipartite element--a phylogenetically conserved stem-loop structure and an AUAUAC sequence, or a part thereof, following this stem-loop. Wild-type capping efficiency was obtained when the AUAUAC motif immediately followed the stem-loop and when the gamma-phosphate of the initiation nucleotide was in close proximity to the capping determinant. Incorporation of a synthetic stem-loop followed by an AUAUAC sequence is sufficient to covert a noncapped heterologous transcript into a capped transcript. Transcripts with the initial 32 nucleotides of Saccharomyces cerevisiae U6 snRNA are accurately capped in HeLa cell extract, indicating that capping machinery from HeLa cells can cap U6 snRNA from an evolutionarily distant eucaryote. The U6-snRNA-specific capping is unusual in that it is RNA sequence dependent, while the capping of mRNAs and other U snRNAs is tightly coupled to transcription and is independent of the RNA sequence.

Base Sequence↗