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PubMed1982

Ribosome structure. Localization of 7-methylguanosine in the small subunits of Escherichia coli and chloroplast ribosomes by immunoelectron microscopy.

The minor nucleoside 7-methylguanosine occurs in Escherichia coli 16 S ribosomal RNA at a single site. High pressure liquid chromatographic analysis shows that a single residue of 7-methylguanosine is also present in chloroplast 16 S ribosomal RNA, presumably at an analogous position in the sequence. Antibodies to 7-methylguanosine were induced in rabbits and shown to be highly specific for the intact methylated base. These antibodies were reacted with 30 S ribosomal subunits from E. coli and from the chloroplasts of Alaskan peas. These two types of ribosome have been shown to be topographically similar (Trempe, M. R., and Glitz, D. G. (1981) J. Biol. Chem. 256, 11873-11879). Electron microscopy of the subunit-antibody complexes showed similar subunit-IgG monomers and antibody-linked subunit dimers. In greater than 95% of the complexes observed for each type of ribosome, antibody contact was consistent with a single binding site, which places 7-methylguanosine near the junction of the upper one-third and lower two-thirds of the subunit and maximally distant from the platform. The analogous localization in both E. coli and chloroplast 30 S ribosomal subunits lends support to their proposed common evolutionary origin.

Antigen-Antibody Complex↗
PubMedDate not supplied

[Age-dependent gene induction in the quail oviduct. Role of post-transcriptional changes in the aging process].

In 1974 we introduced the quail oviduct system to molecular gerontology as a model for investigating age-dependent gene realization at the molecular level. During ageing the amounts of synthesized ovalbumin and avidin, the two major oviduct proteins, decrease to reach almost 0-values. Soon it emerged that the reasons for the reduced ovalbumin and avidin syntheses were not only on the transcriptional but in addition on the posttranscriptional level. It was found that the observed reduced capacity for protein biosynthesis was an age-correlated impairment of those enzyme systems which are involved in the processing of hnRNA to mRNA and in the transport of mRNA from nucleus to cytoplasm. After having firstly discovered and purified some key enzymes of mRNA processing ( endoribonucleases IV, V and VII; U1- snRNP -associated endoribonuclease; poly(A)-specific exoribonuclease ), and secondly studied in detail the enzymically controlled nucleocytoplasmic mRNA transport system, we observed that polyadenylation and perhaps also splicing of hnRNA (two processes in posttranscriptional modification of mRNA) are impaired during ageing. Pronounced changes occur especially at the level of polyadenylation of hnRNA; analytic and enzymic studies revealed that, in parallel with ageing, poly(A) catabolism increases although poly(A) anabolism remains unchanged. The consequences of this change are: reduced stability of mRNA, probably reduced splicing efficiency, reduced binding of mRNA to the inner nuclear membrane or lamina, and reduced nucleocytoplasmic transport of mRNA. The data suggest that changes at the posttranscriptional level are as important in ageing as changes in transcription, and they may explain the observed, age-dependent reduction of the amount of functional mRNA in the cytoplasm.

Aging↗
PubMed1984

[Cleavage of tRNA and rRNA at 7-methylguanine in the presence of methylated carrier RNA].

A method of site-specific cleavage of some tRNAs and rRNAs at the 7-methylguanine residue is described. After reduction of 7-methylguanine by sodium borohydride treatment in the presence of the exogenous carrier RNA methylated statistically by dimethylsulfate the polynucleotide chain is cut at the modified residue by aniline. It was shown that the previously used procedure which did not involve a methylated carrier RNA is not applicable for splitting rRNAs or low concentrations of tRNAs. The method described was successfully used for site-specific cleavage of 32P-terminally labelled yeast tRNAPhe and unlabelled E. coli 16S rRNA at 7-methylguanine position.

Aniline Compounds↗
PubMedDate not supplied

[Processing of tRNA].

The article reviews various aspects of tRNA biosynthesis in pro- and eukaryotes. Some data on the tRNA gene localization are presented. Structures of tRNA precursors are given; their conversion into mature tRNA molecules and the corresponding enzymatic systems are discussed in detail. Special emphasis is given to the transcription of tRNA genes and processing of tRNA precursors in vitro. Similarities and diversities of tRNA processing in pro- and eukaryotes are discussed.

Animals↗
PubMed1994

Position and orientation independent transactivation by c-Myc.

The c-myc oncogene c-Myc is commonly activated in cancer and transactivates gene expression by binding to CACGTG DNA sequences as a heterodimeric complex with Max. The ornithine decarboxylase (ODC), p53, prothymosin alpha and ECA39 promoters are transactivated by c-Myc, and are considered direct targets, as activation is mediated by CACGTG sequences. Interestingly, the c-Myc-responsive CACGTG sequences in the p53, prothymosin alpha, ECA39 and murine ODC genes are all downstream of the RNA CAP site, suggesting that downstream sequences are preferred c-Myc targets. Using a series of heterologous reporter constructs, we have tested the effects of position and orientation of c-Myc-responsive CACGTG sequences on c-Myc's ability to activate transcription. A single binding site conferred c-Myc-responsiveness independent of position and orientation, and over distances of 1.7 kbp. The extent of transactivation was not significantly influenced by position of the responsive elements. By contrast, the extent of transactivation was dependent upon the number of c-Myc binding sites. The results demonstrate that c-Myc activates transcription independent of position and orientation and that considerable flexibility exists in the interaction of c-Myc transactivation domains with the general transcription machinery.

3T3 Cells↗
PubMed1996

Localization of modified nucleotides in Schizosaccharomyces pombe spliceosomal small nuclear RNAs: modified nucleotides are clustered in functionally important regions.

The specific and dynamic RNA:RNA interactions between pre-mRNA and small nuclear RNAs (snRNAs), especially U2, U5, and U6 snRNAs, form the catalytic core and are at the heart of the spliceosome formation. The functionally important regions in the snRNAs correspond to the highly modified regions in snRNAs from human, rat, and plant cells. To better understand the importance of the modifications of snRNAs, we identified and localized the modified nucleotides in the five spliceosomal snRNAs of Schizosaccharomyces pombe cells. Twenty-two modified nucleotides, including base methylations, 2'-O-methylations, and pseudouridines, were found in the five spliceosomal snRNAs. The conservation of modified nucleotides between human and S. pombe snRNAs is striking. In addition, most of the modified nucleotides are in or around positions that form hydrogen bonds with the pre-mRNA or with other snRNAs. The results are consistent with the suggestion that modified nucleotides are clustered around functionally important regions of the spliceosomal snRNAs. These data provide the basis for further functional studies on posttranscriptional modifications in spliceosomal snRNAs.

Base Sequence↗
PubMed1996

Spliced leader RNA of trypanosomes: in vivo mutational analysis reveals extensive and distinct requirements for trans splicing and cap4 formation.

In trypanosomes mRNAs are generated through trans splicing. The spliced leader (SL) RNA, which donates the 5'-terminal mini-exon to each of the protein coding exons, plays a central role in the trans splicing process. We have established in vivo assays to study in detail trans splicing, cap4 modification, and RNP assembly of the SL RNA in the trypanosomatid species Leptomonas seymouri. First, we found that extensive sequences within the mini-exon are required for SL RNA function in vivo, although a conserved length of 39 nt is not essential. In contrast, the intron sequence appears to be surprisingly tolerant to mutation; only the stem-loop II structure is indispensable. The asymmetry of the sequence requirements in the stem I region suggests that this domain may exist in different functional conformations. Second, distinct mini-exon sequences outside the modification site are important for efficient cap4 formation. Third, all SL RNA mutations tested allowed core RNP assembly, suggesting flexible requirements for core protein binding. In sum, the results of our mutational analysis provide evidence for a discrete domain structure of the SL RNA and help to explain the strong phylogenetic conservation of the mini-exon sequence and of the overall SL RNA secondary structure; they also suggest that there may be certain differences between trans splicing in nematodes and trypanosomes. This approach provides a basis for studying RNA-RNA interactions in the trans spliceosome.

Animals↗
PubMed1982

Capping of Viral RNA in Cultured Spodoptera frugiperda Cells Infected with Autographa californica Nuclear Polyhedrosis Virus.

Viral RNA from fall armyworm (Spodoptera frugiperda) cells infected with Autographa californica nuclear polyhedrosis virus contains cap structures. Most of the cap labeled in vivo with [(3)H]methionine or (32)P(i) cochromatographed on DEAE-cellulose with the -5 charge marker; a minor component appeared at -4 net charge. The former is probably a cap 1 structure (m(7)GpppX(m) (p)Yp), and the latter is probably a cap 0 (m(7)GpppXp). On the basis of relative labeling of the two caps with [(3)H]adenosine and [(3)H]guanosine, we concluded that each cap contained at least one adenosine residue in addition to guanosine and, therefore, that cap 0 contained m(7)GpppAp. Cleavage of [(3)H]methionine-labeled viral RNA with tobacco acid pyrophosphatase released a labeled component that cochromatographed on polyethyleneimine-cellulose with m(7)Gp, confirming the m(7)GpppX linkage in the cap. These results were also seen with host polyadenylated RNA. The caps appeared in nearly equal abundance in viral polyadenylated and non-polyadenylated RNAs. The ratio of (32)P(i) incorporated into the cap to that incorporated into mononucleotides suggested average lengths for the polyadenylated and non-polyadenylated RNAs of 1,800 and 1,200 nucleotides, respectively.

Journal Article↗
PubMed1999

Rotavirus open cores catalyze 5'-capping and methylation of exogenous RNA: evidence that VP3 is a methyltransferase.

Rotavirus open cores prepared from purified virions consist of three proteins: the RNA-dependent RNA polymerase, VP1; the core shell protein, VP2; and the guanylyltransferase, VP3. In addition to RNA polymerase activity, open cores have been shown to contain a nonspecific guanylyltransferase activity that caps viral and nonviral RNAs in vitro. In this study, we examined the structure of RNA caps made by open cores and have analyzed open cores for other capping-related enzymatic activities. Utilizing RNase digestion and thin-layer chromatography, we found that the majority ( approximately 70%) of caps made by open cores contain the tetraphosphate linkage, GppppG, rather than the triphosphate linkage, GpppG, found on mRNAs made by rotavirus double-layered particles. Enzymatic analysis indicated that the GppppG caps resulted from the lack of a functional RNA 5'-triphosphatase in open cores, to remove the gamma-phosphate from the RNA prior to capping. RNA 5'-triphosphatases commonly exhibit an associated nucleoside triphosphatase activity, and this too was not detected in open cores. Caps of some RNAs contained an extra GMP moiety (underlined) and had the structure 3'-GpGp(p)ppGpGpC-RNA-3'. The origin of the extra GMP is not known but may reflect the cap serving as a primer for RNA synthesis. Methylated caps were produced in the presence of the substrate, S-adenosyl-l-methionine (SAM), indicating that open cores contain methyltransferase activity. UV cross-linking showed that VP3 specifically binds SAM. Combined with the results of earlier studies, our results suggest that the viral guanylyltransferase and methyltransferase are both components of VP3 and, therefore, that VP3 is a multifunctional capping enzyme.

Acid Anhydride Hydrolases↗
PubMed2006

Amino acid residues in the N-terminal region of the PA subunit of influenza A virus RNA polymerase play a critical role in protein stability, endonuclease activity, cap binding, and virion RNA promoter binding.

The RNA-dependent RNA polymerase of influenza virus is a heterotrimer formed by the PB1, PB2, and PA subunits. Although PA is known to be required for polymerase activity, its precise role is still unclear. Here, we investigated the function of the N-terminal region of PA. Protease digestion of purified recombinant influenza virus A/PR/8/34 PA initially suggested that its N-terminal region is folded into a 25-kDa domain. We then systematically introduced point mutations into evolutionarily conserved amino acids in the N-terminal region of influenza virus A/WSN/33. Most alanine-scanning mutations between residues L109 and F117 caused PA degradation, mediated by a proteasome-ubiquitin pathway, and as a consequence interfered with polymerase activity. Three further PA mutations, K102A, D108A, and K134A, were investigated in detail. Mutation K102A caused a general decrease both in transcription and replication in vivo, whereas mutations D108A and K134A selectively inhibited transcription. Both the D108A and K134A mutations completely inhibited endonuclease activity in vitro, explaining their selective defect in transcription. K102A, on the other hand, resulted in a significant decrease in both cap binding and viral RNA promoter-binding activity and consequently inhibited both transcription and replication. These results suggest that the N-terminal region of PA is involved in multiple functions of the polymerase, including protein stability, endonuclease activity, cap binding, and promoter binding.

Amino Acid Sequence↗
PubMed2004

A function of yeast mRNA cap methyltransferase, Abd1, in transcription by RNA polymerase II.

Capping enzymes bind the phosphorylated pol II CTD permitting cotranscriptional capping of nascent pre-mRNAs. We asked whether these interactions influence pol II function using ChIP in ts mutants of yeast capping enzymes. Pol II occupancy at the 5' ends of PGK1, ENO2, GAL1, and GAL10 was reduced by inactivation of the methyltransferase, Abd1, but not the guanylyltransferase, Ceg1, suggesting that Abd1 contributes to stable promoter binding. At other genes, Abd1 inactivation increased the 5':3' ratio of pol II density in the promoter-proximal region and caused Ser5 hyperphosphorylation of the pol II CTD. These results suggest an additional role for Abd1 in the promoter clearance and/or promoter-proximal elongation steps of transcription. The transcriptional functions of Abd1 are independent of methyltransferase activity. Manipulation of transcription by Abd1 may enhance cotranscriptional capping and also act as a checkpoint to ensure that a nascent transcript has a cap before it can be completed.

Binding Sites↗
PubMed2001

Recombinant hepatitis E virus genomes infectious for primates: importance of capping and discovery of a cis-reactive element.

Hepatitis E virus recombinant genomes transcribed in vitro from two cDNA clones differing by two nucleotides were infectious for chimpanzees. However, one cDNA clone encoded a virus that was attenuated for chimpanzees and unable to infect rhesus monkeys. The second cDNA clone encoded a virus that infected both chimpanzees and rhesus monkeys and caused acute hepatitis in both. One mutation differentiating the two clones identified a cis-reactive element that appeared to overlap the 3' end of the capsid gene and part of the 3' noncoding region. Capping of the RNA transcripts was essential for infectivity.

Animals↗
PubMed1996

A multi-functional enzyme with RNA polymerase and RNase activities: molecular anatomy of influenza virus RNA polymerase.

Influenza virus-associated RNA polymerase is composed of one molecule each of three viral P proteins and carries the complete activity of capped RNA-primed vRNA-directed transcription. The RNA polymerase holoenzyme also carries capped RNA endonuclease to generate capped oligonucleotide primers for transcription and 3'-to-5' exonuclease to remove erroneously polymerized nucleotides at nascent RNA 3' termini prior to the addition of correct substrates. PB1 is the core subunit for not only RNA synthesis but also the assembly of PB2 and PA into the holoenzyme complex, while PB2 plays a key role in capped RNA cleavage. The transcriptase is converted into the RNA replicase with the full activity of replication, ie vRNA-directed cRNA synthesis and cRNA-directed vRNA synthesis, after interaction with an as yet unidentified host factor(s).

DNA-Directed RNA Polymerases↗
PubMed1984

Chemical synthesis of the 5'-terminal part bearing cap structure of messenger RNA of cytoplasmic polyhedrosis virus (CPV): m7G5'pppAmpG and m7G5'pppAmpGpU.

The 5'-terminal structures of mRNA bearing the so-called 'cap' from cytoplasmic polyhedrosis virus (CPV), m7G5' pppAmpG and m7G5' pppAmpGpU, were first chemically synthesized. S,S-Di(4-methoxyphenyl) N6-benzoyl-2'-O-methyladenosine 5'-phosphorodithioate ((ArS) 2pAbmz) was prepared by phosphorylation of the 5'-hydroxyl group of N6-benzoyl-2'-O-methyladenosine with S,S-di(4-methoxyphenyl) phosphorodithioate by TPS. By the triester approach using (ArS) 2pAbmz as starting material, the protected dinucleotide and trinucleotide bearing 5'-phosphate group were synthesized. The protective groups of the dinucleotide and trinucleotide were removed to obtain pAmpG and pAmpGpU, respectively. By the reaction of a capping agent ((PhS) ppm7G) with pAmpG and pAmpGpU in the presence of silver nitrate or iodine. The 5'-terminal structure of the messenger RNA strand of CPV which was labelled isotopically, was confirmed completely as m7G5' pppAmGpU by cochromatography with the materials chemically synthesized here.

Chemical Phenomena↗
PubMed1992

Prokaryotic-like cis elements in the cap-independent internal initiation of translation on picornavirus RNA.

Initiation of translation on picornavirus RNAs is accomplished through internal binding of ribosomes to a complex cis-acting element. Here we show that efficient function of this element involves two appropriately spaced smaller elements: UUUCC and an AUG. This conclusion emerged from analysis of the genome structures of spontaneous revertants of mutant polioviruses with extended insertions between the UUUCC and AUG motifs. It was confirmed by the results obtained with specially designed constructs. A similarity to the prokaryotic translation initiation mechanism, which involves the Shine-Dalgarno sequence, is emphasized, but in the picornavirus system the position of the UUUCC must be strictly fixed relative to upstream cis-acting elements, and the AUG may not necessarily serve as an initiation codon.

Base Sequence↗
PubMed1997

[Transcription and replication of influenza virus genome].

The genome of influenza virus is composed of eight RNA segments of negative polarity. The RNA-dependent RNA polymerase is associated with each viral RNA (vRNA) segment and after infection, involved in both transcription (vRNA-directed synthesis of viral mRNA) and vRNA replication (vRNA-dependent synthesis of complementary RNA(cRNA) and cRNA-dependent synthesis of vRNA). The RNA polymerase is composed of three viral proteins, PB1, PB2 and PA. PB1 is the core subunit for not only the RNA synthesis but also the assembly of PB2 and PA into this multifunctional enzyme complex. PB1 alone is able to catalyze vRNA-dependent RNA synthesis, but PB2 is required for capped RNA-dependent transcription, both together forming the transcriptase. The third P protein, PA, and an as yet unidentified host factor(s) are involved for the conversion of RNA polymerase from transcriptase to replicase. The functional map including both subunit-subunit contact sites and catalytic sites for capped RNA endonuclease and RNA polymerization is being made for both PB1 and PB2 proteins.

Genome, Viral↗