Showing papers on "RNA published in 1988"
TL;DR: HIV-1 encodes a potent trans-activator protein, tat, which is essential for viral gene expression, and chemically synthesized the 86 amino acid tat protein (tat-86) and tat mutant peptides, demonstrating the functional significance of these domains.
1,446 citations
TL;DR: In vitro mutagenesis of sequences required for the self-catalysed cleavage of a plant virus satellite RNA has allowed definition of an RNA segment with endoribonuclease activity.
Abstract: In vitro mutagenesis of sequences required for the self-catalysed cleavage of a plant virus satellite RNA has allowed definition of an RNA segment with endoribonuclease activity. General rules have been deduced for the design of new RNA enzymes capable of highly specific RNA cleavage, and have been successfully tested against a new target sequence.
1,383 citations
TL;DR: In this paper, the effect of the poliovirus 5'NTR on in vitro translation was compared with that of the encephalomyocarditis virus by using synthetic mRNAs, micrococcal nuclease-treated HeLa cell extracts, and rabbit reticulocyte lysates.
Abstract: Picornavirus RNAs are uncapped messengers and have unusually long 5' nontranslated regions (5'NTRs) which contain many noninitiating AUG triplets. The translational efficiency of different picornavirus RNAs varies between different cell-free extracts and even in the same extract, such as micrococcal nuclease-treated rabbit reticulocyte lysates. The effect of the poliovirus 5'NTR on in vitro translation was compared with that of the 5'NTR of encephalomyocarditis virus by the use of synthetic mRNAs, micrococcal nuclease-treated HeLa cell extracts, and rabbit reticulocyte lysates. Artificial mono- and dicistronic mRNAs synthesized with T7 RNA polymerase were used to investigate whether the 5'NTR of encephalomyocarditis virus RNA contains a potential internal ribosomal entry site. The sequence between nucleotides 260 and 484 in the 5'NTR of encephalomyocarditis RNA was found to play a critical role in the efficient translation in both mono- and dicistronic mRNAs. Our data suggest that an internal ribosomal entry site resides in this region.
1,363 citations
TL;DR: The predicted carboxy-terminal protein sequence encoded by the pNM23 cDNA clone is novel compared with Genebank animal, bacterial, and viral sequences and may be associated with intrinsic aggressiveness.
Abstract: We describe a gene, NM23, that is associated with the tumor metastatic process. NM23 RNA levels were highest in cells and tumors of relatively low metastatic potential in two experimental systems: (1) murine K-1735 melanoma cell lines, in which the gene was identified, and (2) N-nitroso-N-methylurea-induced rat mammary carcinomas. NM23 RNA levels did not correlate with cell sensitivity to host immunological responses and may, therefore, be associated with intrinsic aggressiveness. The predicted carboxy-terminal protein sequence encoded by the pNM23 cDNA clone is novel compared with Genebank animal, bacterial, and viral sequences.
1,283 citations
TL;DR: Macrophages isolated from a wound site, and not exposed to cell culture conditions, expressed messenger RNA transcripts for TGF-alpha, T GF-beta, platelet-derived growth factor A-chain, and insulin-like growth factor-1 by a novel method for RNA analysis.
Abstract: The presence of macrophages is required for the regeneration of many cell types during wound healing. Macrophages have been reported to express a wide range of mitogenic factors and cytokines, but none of these factors has been shown in vivo to sustain all the wound-healing processes. It has been suggested that transforming growth factor-alpha (TGF-alpha) may mediate angiogenesis, epidermal regrowth, and formation of granulation tissue in vivo. Macrophages isolated from a wound site, and not exposed to cell culture conditions, expressed messenger RNA transcripts for TGF-alpha, TGF-beta, platelet-derived growth factor A-chain, and insulin-like growth factor-1. The expression of these transcripts was determined by a novel method for RNA analysis in which low numbers of mouse macrophages were isolated from wound cylinders, their RNA was purified and reverse-transcribed, and the complementary DNA was amplified in a polymerase chain reaction primed with growth factor sequence-specific primers. This single-cell RNA phenotyping procedure is rapid and has the potential for quantification, and mRNA transcripts from a single cell or a few cells can be unambiguously demonstrated, with the simultaneous analysis of several mRNA species. Macrophages from wounds expressed TGF-alpha antigen, and wound fluids contained TGF-alpha. Elicited macrophages in culture also expressed TGF-alpha transcripts and polypeptide in a time-dependent manner after stimulation with modified low-density lipoproteins and lipopolysaccharide endotoxin, which are characteristic of the activators found in injured tissues.
1,136 citations
TL;DR: It is reported that translation of HIV-1 RNA synthesized in vitro by SP6 RNA polymerase yields significant amounts of a gag-pol fusion protein, indicating that efficient ribosomal frameshifting also occurs within the HIV--pol overlap region.
Abstract: Based on precedents from other retroviruses, the precursor of the human immunodeficiency virus (HIV-1) reverse transcriptase is predicted to be a polyprotein with a relative molecular mass (Mr) of 160,000 (160K) encoded by both the viral pol gene and the upstream gag gene. These two genes lie in different translational reading frames, with the 3' end of gag overlapping the 5' end of pol by 205 or 241 nucleotides. Thus, production of the gag-pol fusion protein would require either messenger RNA processing or translational frameshifting. The latter mechanism has been shown in the synthesis of the gag-pol proteins of two other retroviruses, Rous sarcoma virus (RSV) and mouse mammary tumour virus (MMTV). Here we report that translation of HIV-1 RNA synthesized in vitro by SP6 RNA polymerase yields significant amounts of a gag-pol fusion protein, indicating that efficient ribosomal frameshifting also occurs within the HIV-1 gag-pol overlap region. Site-directed mutagenesis and amino-acid sequencing localized the site of frameshifting to a UUA leucine codon near the 5' end of the overlap.
969 citations
TL;DR: The kinetics of induction after serum starvation and density-dependent inhibition of two growth-arrest-specific (gas) genes were investigated in more detail and two cell lines transformed by viral onc genes did not express the two gas genes.
904 citations
TL;DR: Results indicate that both VT2 and Shiga toxin inactivate 60S ribosomal subunits by cleaving the N-glycosidic bond at A-4324 in 28S ribOSomal RNA.
Abstract: The site of action of a Vero toxin (VT2 or Shiga-like toxin II) from enterohemorrhagic Escherichia coli and Shiga toxin from Shigella dysenteriae 1 on eukaryotic ribosomes was studied. Treatment of eukaryotic ribosomes with either toxin caused the release of a fragment of 400 nucleotides from 28S ribosomal RNA when the isolated ribosomal RNA was treated with aniline. Release of this fragment with aniline treatment was accompanied by inhibition of protein synthesis and of elongation-factor-1-dependent aminoacyl-tRNA binding to ribosomes. Analysis of the nucleotide sequence of the 3'-terminal fragment of 553 nucleotides of 28S rRNA of rat liver 60S ribosomal subunits suggested that an adenine base at position 4324 (A-4324) was absent in toxin-treated 28S rRNA. Further analysis by thin-layer chromatography demonstrated quantitative release of adenine from rat liver ribosomes on treatment with the toxins. These results indicate that both VT2 and Shiga toxin inactivate 60S ribosomal subunits by cleaving the N-glycosidic bond at A-4324 in 28S ribosomal RNA.
779 citations
TL;DR: It is demonstrated that during the reaction many, but not all, of the adenosine residues are converted to inosine residues, and it is proposed that the covalent modification is responsible for the irreversible change in base pairing properties.
657 citations
TL;DR: It is suggested that the 3′ AU-rich sequences act to destabilize the mRNA by directing rapid removal of the mRNA poly(A) tract.
Abstract: The c-fos proto-oncogene provides a good system to study the processes underlying messenger RNA degradation. After growth factor stimulation of susceptible cells, the c-fos transcription rate transiently increases from a low basal level by as much as 50-fold, producing a large amount of exceedingly unstable c-fos mRNA that is rapidly degraded. Here, we investigate the c-fos mRNA degradation process, and find that: (1) ongoing translation of the c-fos mRNA itself is required for its degradation; (2) after synthesis, the mRNA poly(A) tail is rapidly removed, in a translation-dependent manner, leading to accumulation of apparently deadenylated RNA; (3) deletion or replacement of an AU-rich sequence at the mRNA 3' end significantly stabilizes the mRNA; (4) deletion of the 3' AU-rich sequences dramatically slows the poly(A) shortening rate. These results suggest that the 3' AU-rich sequences act to destabilize the mRNA by directing rapid removal of the mRNA poly(A) tract.
656 citations
TL;DR: A cell-free translation system has been constructed that uses a continuous flow of the feeding buffer through the reaction mixture and a continuous removal of a polypeptide product, and has proven active for long times.
Abstract: A cell-free translation system has been constructed that uses a continuous flow of the feeding buffer [including amino acids, adenosine triphosphate (ATP), and guanosine triphosphate (GTP)] through the reaction mixture and a continuous removal of a polypeptide product. Both prokaryotic (Escherichia coli) and eukaryotic (wheat embryos, Triticum sp.) versions of the system have been tested. In both cases the system has proven active for long times, synthesizing polypeptides at a high constant rate for tens of hours. With the use of MS2 phage RNA or brome mosaic virus RNA 4 as templates, 100 copies of viral coat proteins per RNA were synthesized for 20 hours in the prokaryotic or eukaryotic system, respectively. With synthetic calcitonin messenger RNA, 150 to 300 copies of calcitonin polypeptide were produced per messenger RNA in both types of continuous translation systems for 40 hours.
TL;DR: A short sequence of RSV RNA, 147 nucleotides in length, containing the frameshift site and stem-loop structure, is sufficient to direct frameshifting in a novel genetic context.
TL;DR: The design of the method and careful optimization of first strand synthesis have permitted cloning of several large cDNAs, and Transcription of essentially full-length clones with phage SP6 RNA polymerase produces RNAs that are efficiently translated in vitro to give complete, unfused products, thus permitting rapid characterization of the clones via the encoded polypeptides.
TL;DR: Analysis of the changes in transcription and mRNA levels after serum stimulation demonstrated that the kinetics and extent of the induction vary dramatically between the different genes, and showed that induction of proliferation is accompanied by the onset of a complex genetic program.
Abstract: Genes whose expression is growth factor regulated are likely to be important components in the mechanisms controlling cell proliferation and differentiation. With the aim of identifying some of those genes, a lambda cDNA library was prepared with poly(A)+ RNA from quiescent NIH 3T3 cells stimulated with serum for 4 h in the presence of cycloheximide. Differential screening of approximately 200,000 recombinant phage plaques revealed 2,540 clones that cross hybridized preferentially with [32P]cDNA derived from RNA of stimulated cells rather than with cDNA derived from nonstimulated cells. Cross hybridization of these clones identified 82 independent sequences, including c-fos and c-myc. Seventy-one clones were further studied. Analysis of the changes in transcription and mRNA levels after serum stimulation demonstrated that the kinetics and extent of the induction vary dramatically between the different genes. Cycloheximide in all cases superinduced the mRNA levels by two mechanisms, inhibiting the shutoff of transcription and prolonging the half-lives of the mRNAs. Our results showed that induction of proliferation is accompanied by the onset of a complex genetic program.
TL;DR: Analysis of a series of mutants of an Escherichia coli alanine transfer RNA shows that substitution of a single G-U base pair in the acceptor helix eliminates aminoacylation withAlanine in vivo and in vitro.
Abstract: Analysis of a series of mutants of an Escherichia coli alanine transfer RNA shows that substitution of a single G-U base pair in the acceptor helix eliminates aminoacylation with alanine in vivo and in vitro. Introduction of that base pair into the analogous position of a cysteine and a phenylalanine transfer RNA confers upon each the ability to be aminoacylated with alanine. Thus, as little as a single base pair can direct an amino acid to a specific transfer RNA.
TL;DR: The results of nucleotide substitution experiments are presented which suggest that tat trans-activation requires presentation of the sequence +30CUGGG+34 in tar within the loop of a RNA hairpin structure.
Abstract: Human immunodeficiency virus (HIV-1) is the primary retroviral agent responsible for AIDS and related disorders worldwide. One of its identified gene products, tat protein, stimulates in trans the expression of all HIV-1 genes by several orders of magnitude1–7. Cells infected with HIV-1 require tat protein to produce virus, suggesting that trans-activation is crucial for viral replication8,9. The essential cis-acting site for trans-activation, termed tar, resides within the R region of the HIV-1 long terminal repeat (LTR), between −17 and +54 with respect to the initiation site of viral transcription10,11. It is striking that the RNA encoded between +1 and +59 has the potential to form an extensive stem-loop secondary structure which, as a portion of the untranslated leader RNA, would be common to all HIV-1 mRNAs 11,12. We now present the results of nucleotide substitution experiments which suggest that tat trans-activation requires presentation of the sequence +30CUGGG+34 in tar within the loop of a RNA hairpin structure.
TL;DR: In situ hybridization with specific complementary DNA probes are used to show that messenger RNA for the dendrite-specific microtubule-associated protein MAP2 is present in dendrites in the developing brain.
Abstract: For nerve cells to develop their highly polarized form, appropriate structural molecules must be targeted to either axons or dendrites. This could be achieved by the synthesis of structural proteins in the cell body and their sorting to either axons or dendrites by specific transport mechanisms. For dendrites, an alternative possibility is that proteins could be synthesized locally in the dendritic cytoplasm. This is an attractive idea because it would allow regulation of the production of structural molecules in response to local demand during dendritic development. The feasibility of dendritic protein synthesis is suggested both by the existence of dendritic polyribosomes1 and by the recent demonstration that newly synthesized RNA is transported into the dendrites of neurons differentiating in culture2. However, to date there has been no demonstration of the selective synthesis of an identified dendrite-specific protein in the dendritic cytoplasm. Here, we use in situ hybridization with specific complementary DNA probes to show that messenger RNA for the dendrite-specific microtubule-associated protein MAP2 (refs 3–5) is present in dendrites in the developing brain. By contrast the mRNA for tubulin, a protein present in both axons and dendrites4,6,7 is located exclusively in neuronal cell bodies.
TL;DR: The 3' untranslated region of hTR cDNA was sufficient to confer iron-dependent regulation on another gene, and the regulated phenotype correlates with the presence of a stem-loop structure predicted by a computer algorithm.
TL;DR: The RNA genome of the cytopathic NADL isolate of bovine viral diarrhea virus has been molecularly cloned and the nucleotide sequence determined and a single major open reading frame was found in the viral-sense (positive polarity) sequence.
TL;DR: A cDNA clone was isolated, MK1, whose RNA level increased in early stages of retinoic acid-induced differentiation of embryonal carcinoma cells, and the amount of MK1 RNA progressively decreased in the later stages of the differentiation.
TL;DR: The authors used chemical probes to investigate the possible interaction of the elongation factors EF-Tu and EF-G with ribosomal RNA in E. coli ribosomes, and observed EFG-dependent footprints in vitro and in vivo around position 1,067 in domain II of 23S rRNA, and in the loop around position 2,660 in domain VI.
Abstract: The elongation factors EF-Tu and EF-G interact with ribosomes during protein synthesis: EF-Tu presents incoming aminoacyl transfer RNA to the programmed ribosome as an EF-Tu-GTP-tRNA ternary complex and EF-G promotes translocation of peptidyl-tRNA and its associated messenger RNA from the A to the P site after peptidyl transfer. Both events are accompanied by ribosome-dependent GTP hydrolysis. Here we use chemical probes to investigate the possible interaction of these factors with ribosomal RNA in E. coli ribosomes. We observe EF-G-dependent footprints in vitro and in vivo around position 1,067 in domain II of 23S rRNA, and in the loop around position 2,660 in domain VI.EF-Tu gives an overlapping footprint in vitro at positions 2,655 and 2,661, but shows no effect at position 1,067. The 1,067 region is the site of interaction of the antibiotic thiostrepton, which prevents formation of the EF-G-GTP-ribosome complex and is a site for interaction with the GTPase-related protein L11 (ref. 3). The universally conserved loop in the 2,660 region is the site of attack by the RNA-directed cytotoxins alpha-sarcin and ricin, whose effects abolish translation and include the loss of elongation factor-dependent functions in eukaryotic ribosomes.
Book•
01 Apr 1988
TL;DR: This work has shown clear trends in the recruitment and degradation of U-snRNPs towards ‘spiking’ in the presence of ribonuclease P, and these trends are likely to be related to each other.
Abstract: Small Nuclear RNAs: RNA Sequences, Structure, and Modifications.- The Genes and Transcription of the Major Small Nuclear RNAs.- snRNP Proteins.- UsnRNP Assembly and Transport.- Functions of the Abundant U-snRNPs.- Structure and Function of Minor snRNPs.- Ribonuclease P as a snRNP.- Genetic Analysis of Yeast snRNAs.
TL;DR: Sequence elements conserved among the available set of 66 group I introns are compiled and the possible significance of conserved nucleotides within base-paired duplexes is discussed.
TL;DR: It is proposed that Sxl encodes a factor that interacts with both its own pre-mRNA and that of downstream genes to confer female-specific splicing, so that a single, simple mechanism could account for both the maintenance and expression of the sexually determined state.
TL;DR: An extremely simple procedure for preparing cytoplasmic RNA from small numbers of cells is described and RNA is ready for Northern gel analysis in less than 30 min.
TL;DR: Both the codon and amino-acid specificity of this tRNA are converted by a single post-transcriptional modification of the first position of the anticodon during tRNA maturation.
Abstract: An Escherichia coli isoleucine transfer RNA specific for the codon AUA (tRNA2Ile or tRNAminorIle (ref. 1) has a novel modified nucleo-side, lysidine (L; ref. 2) (Fig. la) in the first position of the anticodon (position 34), which is essential for the specific recognition of the codon AUA (ref. 1). We isolated the gene for tRNA2Ile (ileX) and found that the anticodon is CAT, which is characteristic of the methionine tRNA gene. Replacement of L(34) of tRNA2Ile molecule enzymatically with unmodified C(34) resulted in a marked reduction of the isoleucine-accepting activity and, surprisingly, in the appearance of methionine-accepting activity. Thus, both the codon and amino-acid specificity of this tRNA are converted by a single post-transcriptional modification of the first position of the anticodon during tRNA maturation.
TL;DR: This chapter focuses on the structural analysis of RNA using chemical and enzymatic probing monitored by primer extension, and Dimethyl sulfate and l-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho- p -toluene sulfonate (CMCT) are employed for chemical probing, while ribonucleases A and T 1 , and V l nuclease are employed as enzymatics probes.
Abstract: Publisher Summary This chapter focuses on the structural analysis of RNA using chemical and enzymatic probing monitored by primer extension. Chemical and enzymatic probing, monitored by primer extension, has become a powerful tool for the analysis of RNA structure. The reactivities of individual nucleotides composing large RNA molecules may be determined rapidly by utilizing a series of primers spaced at approximately 200 nucleotide intervals. In addition, numerous chemical reagents and nucleases may be employed as probes, since the only requirement is that they modify the template so as to produce pauses or stops in the progress of reverse transcriptase. The RNA, either alone or complexed with proteins and/or ligands, is incubated under suitable conditions with chemical or enzymatic probes. Dimethyl sulfate (DMS), kethoxal (KE) and l-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho- p -toluene sulfonate (CMCT) are employed for chemical probing, while ribonucleases A and T 1 , and V l nuclease are employed as enzymatic probes. The extent of the reactions is limited so that no more than a few stops are present within 300 nucleotide stretches in a given RNA molecule.
TL;DR: Sequences from the transcribed region of the herpes simplex virus thymidine kinase gene, a gene that lacks an intervening sequence, permitted substantial intron-independent expression (greater than 100-fold increase) in the plasmid vector system.
Abstract: Recombinant simian virus 40 viruses carrying rabbit beta-globin cDNA failed to express the beta-globin sequence unless an intron was included in the transcription unit. The addition of either beta-globin IVS1 or IVS2 caused a 400-fold increase in RNA production. Stable beta-globin RNA production required sequences in IVS2 that were very close to the splice sites and that coincided with those needed for mRNA splicing. In addition to the recombinant viruses, intron-dependent expression was observed with both replicating and nonreplicating plasmid vectors in short-term transfections of cultured animal cells. Unlike transcriptional enhancer elements, IVS2 failed to increase stable RNA production when it was placed downstream of the polyadenylation site. Using a plasmid vector system to survey different inserted sequences for their dependence on introns for expression, we found that the presence of IVS2 stimulated the expression of these sequences 2- to 500-fold. Sequences from the transcribed region of the herpes simplex virus thymidine kinase gene, a gene that lacks an intervening sequence, permitted substantial intron-independent expression (greater than 100-fold increase) in the plasmid vector system.
TL;DR: It is demonstrated that a 70S–fMet-tRNA Met f complex, bound to the coat protein initiation site on Q β plus strand RNA, cannot be dislodged by Q β replicase engaged in negative strand synthesis.
Abstract: Publisher Summary This chapter presents extension inhibition to the study of translation initiation complexes. The chapter also describes the method of using bacteriophage T4 gene 32 mRNA. Early experiments showed that ribosomes, in the presence of fMet-tRNA Met f could properly select translation initiation regions of bacteriophage RNA and protect these regions from ribonuclease digestion. This chapter subsequently demonstrated that a 70S–fMet-tRNA Met f complex, bound to the coat protein initiation site on Q β plus strand RNA, cannot be dislodged by Q β replicase engaged in negative strand synthesis. It was reasoned that specific binding of ribosomes (30S or 70S) to mRNA might lead to pausing or termination of reverse transcriptase when a bound ribosome is encountered. In extension inhibition, a 5'- 32 P-end-labeled oligo deoxyribonucleotide, complementary to a region on the mRNA that is 3' to the initiation codon, is annealed to either total cellular RNA or to a purified transcript. This [ 32 P]oligonucleotide-mRNA hybrid is then incubated with ribosomes, and the complexes analyzed by primer extension.