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PubMed1985

RNA structural dynamics: pre-melting and melting transitions in E. coli 5S rRNA.

The temperature dependent transition from duplex to a single strand in E. coli 5S ribosomal RNA is a multistep process, and it involves intermediate states. We have analyzed these structural dynamics by chemical modification of cytidines and by single strand specific nuclease digestions. This combined approach led to the characterization of premelting and melting transitions within individual structural segments of the native macromolecule, which we feel may find general application to the structure of biological polyribonucleotides: 1) G-C base pairs at the termini of helices are relatively unstable and they readily undergo premelting transition. 2) Internal G-U/A-U rich stretches of helices exhibit dynamic premelting properties. 3) Hairpin loops have a relatively stronger destabilizing effect than internal loops. 4) Bulge loops destabilize the neighbouring base pairs. 5) Melting of helical segments occurs starting from the destabilizing structures listed above, preferentially from the helix termini. E. coli 5S rRNA has been shown to adopt different conformations. The presence of urea leads to induction of enhancement in the sensitivity for nuclease S1 at several nucleotide positions. The possibility of structural rearrangements will be discussed.

Base Sequence↗
PubMed1997

Single-strand-preferring RNases degrade double-stranded RNAs by destabilizing its secondary structure.

To establish the mechanism of dsRNA degradation by mammalian single-stranded-preferring ribonucleases, and, in particular, the influence of their positively charged non-catalytic amino acid residues, we have studied the kinetic parameters of the depolimerization of single- and double-stranded polyribonucleotides such as poly(U), poly(U).poly(A), poly(C) and poly(C).poly(I) by the action of human seminal RNase, bovine seminal RNase and ox pancreas RNase A. While the activities of these RNases on poly(I).poly(C) were definitely lower than those on poly(C), the activities of human seminal and bovine seminal RNases on poly(U).poly(A) and poly(U) were of the same order of magnitude under physiological salt conditions. The ratio of the RNase A degrading activities towards poly(U) and poly(U).poly(A) at I = 0.16 M is ten times higher than the corresponding ratios determined with bovine seminal and human seminal ribonucleases. The high activities of these two RNases towards poly(U).poly(A) are discussed on the basis of their efficient estabilishing action on this double-helical nucleic acid due to their high affinity for poly(A). The destabilizing action of human seminal RNase and bovine seminal RNase on the poly (U).poly(A) duplex is higher than that measurable with bovine RNase A because of the higher number of positive charges present on those enzyme molecules. This may therefore explain why human seminal and bovine seminal ribonucleases are more efficient than RNase A in the depolymerization of poly(U).poly(A) at physiological ionic strength.

Animals↗
PubMed2000

Pseudouridine in RNA: what, where, how, and why.

Pseudouridine (5-ribosyluracil) is a ubiquitous yet enigmatic constituent of structural RNAs (transfer, ribosomal, small nuclear, and small nucleolar). Although pseudouridine (psi) was the first modified nucleoside to be discovered in RNA, and is the most abundant, its biosynthesis and biological roles have remained poorly understood since its identification as a "fifth nucleoside" in RNA. Recently, a combination of biochemical, biophysical, and genetic approaches has helped to illuminate the structural consequences of psi in polyribonucleotides, the biochemical mechanism of U-->psi isomerization in RNA, and the role of modification enzymes (psi synthases) and box H/ACA snoRNAs, a class of eukaryotic small nucleolar RNAs, in the site-specific biosynthesis of psi. Through its unique ability to coordinate a structural water molecule via its free N1-H, psi exerts a subtle but significant "rigidifying" influence on the nearby sugar-phosphate backbone and also enhances base stacking. These effects may underlie the biological role of most (but perhaps not all) of the psi residues in RNA. Certain genetic mutants lacking specific psi residues in tRNA or rRNA exhibit difficulties in translation, display slow growth rates, and fail to compete effectively with wild-type strains in mixed culture. In particular, normal growth is severely compromised in an Escherichia coli mutant deficient in a pseudouridine synthase responsible for the formation of three closely spaced psi residues in the mRNA decoding region of the 23S rRNA. Such studies demonstrate that pseudouridylation of RNA confers an important selective advantage in a natural biological context.

Animals↗
PubMed2005

Confocal microscopy studies of a model oligoribonucleotide HIV inhibitor.

Previous work has shown that novel amphipalhic oligo and polyribonucleotides are potent inhibitors of HIV. It was hypothesized that the mechanism(s) of action for these compounds might be inhibition of retroviral reverse transcriptase (RT) and/or viral uptake by cells. A fluorescent oligonucleotide analog was prepared, and confocal microscopy studies were undertaken in order to examine oligonucleotide-cell interactions.

Anti-HIV Agents↗
PubMed1986

Astrocytes produce interferon that enhances the expression of H-2 antigens on a subpopulation of brain cells.

Using primary culture methods, we show that purified astrocytes from embryonic mouse or rat central nervous system (CNS) can be induced to produce interferon (IFN) activity when pretreated with a standard IFN-superinducing regimen of polyribonucleotide, cycloheximide, and actinomycin D, whereas IFN activity was not inducible in neuronal cultures derived from mouse CNS. Astrocyte IFN displays inductive, kinetic, physicochemical, and antigenic properties similar to those of IFN-alpha/beta, but is dissimilar to lymphocyte IFN (IFN-gamma). Treatment of pure astrocytic cultures or astrocytes cultured with neurons with astrocyte IFN or IFN-alpha/beta induced a dramatic increase in the expression of H-2 antigens on a subpopulation of astrocytes. Neither neurons nor oligodendroglia expressed detectable levels of H-2 antigens when exposed to astrocyte IFN, IFN-alpha/beta, or to IFN-beta. Injection of astrocyte IFN or IFN-alpha/beta directly into brains of newborn mice indicated that H-2 antigens were also induced in vivo. None of the IFNs (astrocyte, alpha/beta, or beta) tested induced Ia antigens on CNS cells in vitro or in vivo. Since H-2 antigens have a critical role in immune responses, astrocyte IFN may initiate and participate in immune reactions that contribute to immunoprotective and immunopathological responses in the CNS.

Animals↗
PubMed1986

Production and characterization of poly(I):poly(C)-induced rat interferons in vitro.

The synthetic polyribonucleotide polyinosinate:polycytidylate [poly(I):poly(C)] was used to induce interferon (IFN) production in a rat leiomyosarcoma cell line. Three separate IFN species were distinguished by antiviral activity on homologous and heterologous cells, interaction with Blue Sepharose CL-6B chromatography matrix, and antibody neutralization studies. Two IFN species, one with a weakly ionic and the other with a hydrophobic interaction with Blue Sepharose, exhibited heterologous antiviral activity, and were acid- and heat-labile. Both had a molecular weight of 30,000 daltons by HPLC-based molecular exclusion chromatography. The weakly ionic species was neutralized only by antiserum to L-cell IFN, whereas the hydrophobic species was neutralized by antiserum to L-cell IFN and antiserum to human leukocyte IFN. The remaining IFN species, with a weakly ionic interaction with Blue Sepharose, was acid- and heat-stable, exhibited no heterologous cell antiviral activity, and was not neutralized by any antisera to IFN. This species had a molecular weight of 39,000 daltons by HPLC-based molecular exclusion chromatography and 34,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These rat fibroblast IFNs possess greater heterogeneity than that reported or observed for murine or human fibroblast IFNs. These IFNs could thus be classified as follows: one species of rat IFN-beta and two different species of unique acid-labile rat IFNs.

Animals↗
PubMed1987

Interferon inducibility and sensitivity of human teratocarcinoma-derived cell lines.

Three cell lines tera I, tera II, and PA1, derived from human teratocarcinomas were tested for their capacity to produce interferon (IFN) and for their sensitivity to both human IFN-alpha and IFN-beta. When treated with Newcastle disease virus or Sendai virus, or a synthetic polyribonucleotide, poly(rI):poly(rC), tera I cells produced no IFN and the 2',5'-oligoadenylate (2-5A) synthetase enzymatic pathway was not activated, although there was an increase in protein kinase. In contrast, tera II and PA1 cells produced IFN and both enzymatic activities were detected. IFN treatment has no effect on the growth of any of the cell lines. Tera I and PA1 cells did not develop resistance to challenge with vesicular stomatitis virus or encephalomyocarditis virus, but the growth of a type-C baboon retrovirus was inhibited. Tera II cells were protected against all three viruses. It appears that human teratocarcinoma cell lines can thus differ greatly in their ability to produce IFN and to respond to it.

2',5'-Oligoadenylate Synthetase↗
PubMed1996

Involvement of interleukin-6 and interferon-alpha in the poly(A).Poly(U)-induced 2',5'-oligoadenylate synthetase activity in the mouse monocyte-macrophage cell line, J774A1.

The synthetic polyribonucleotide poly(A).poly(U) induces 2',5'-oligoadenylate synthetase activity in the murine macrophage cell line J774A1. The possible role of several cytokines involved in macrophage activation (i.e., IL-1, IL-6, TNF, and IFN) was examined in the present study. It was first demonstrated that among the anticytokine antibodies, only monoclonal antibodies directed against IL-6 inhibited the induction of 2',5'-oligoadenylate synthetase by poly(A).poly(U) in a dose-dependent manner. Moreover, it was established that poly(A).poly(U) elicited IL-6 production in J774A1 cells in a time-and dose-dependent manner. Consequently, the effect of IL-6 on 2',5'-oligoadenylate synthetase activity was studied. IL-6 either alone or in combination with IL-1 and TNF did not induce 2',5'-oligoadenylate synthetase activity. IL-6 did not potentiate IFN-gamma-induced 2'-5'-oligoadenylate synthetase activity. In contrast, addition of IL-6 to the incubation medium potentiated the stimulation of 2'-5'-oligoadenylate synthetase activity by IFN-alpha. These results suggest that IL-6 is a necessary but not sufficient factor in the induction of 2'-5'-oligoadenylate synthetase activity in the J774A1 cell line by poly(A).poly(U).

2',5'-Oligoadenylate Synthetase↗
PubMed2001

Functional involvement of G8 in the hairpin ribozyme cleavage mechanism.

The catalytic determinants for the cleavage and ligation reactions mediated by the hairpin ribozyme are integral to the polyribonucleotide chain. We describe experiments that place G8, a critical guanosine, at the active site, and point to an essential role in catalysis. Cross-linking and modeling show that formation of a catalytic complex is accompanied by a conformational change in which N1 and O6 of G8 become closely apposed to the scissile phosphodiester. UV cross-linking, hydroxyl-radical footprinting and native gel electrophoresis indicate that G8 variants inhibit the reaction at a step following domain association, and that the tertiary structure of the inactive complex is not measurably altered. Rate-pH profiles and fluorescence spectroscopy show that protonation at the N1 position of G8 is required for catalysis, and that modification of O6 can inhibit the reaction. Kinetic solvent isotope analysis suggests that two protons are transferred during the rate-limiting step, consistent with rate-limiting cleavage chemistry involving concerted deprotonation of the attacking 2'-OH and protonation of the 5'-O leaving group. We propose mechanistic models that are consistent with these data, including some that invoke a novel keto-enol tautomerization.

Base Sequence↗
PubMed1975

Maturation and differentiation of B16 melanoma cells induced by theophylline treatment.

Recent studies suggested that 3',5'-cyclic AMP (CAMP) may be involved in the regulation of cell proliferation and differentiation. Theophylline, an inhibitor of cyclic nucleotide phosphodiesterase, elevated intracellular cAMP. A melanotic clone of the B16 melanoma was treated with theophylline and studied in vitro and in vivo. With 12 hours after 1.0 mM theophylline was added to growing cultures, the number of cells incorporating tritiated thymidine (3-H-TDR) and the rate of uptake of 3-H-TDR into DNA were significantly reduced. After 7 days, the number of cells in the control cultures increased twenty-four times, whereas theophylline-treated cells increased only sixfold. Compared to the controls, the theophylline-treated cells contained ten times the melanin and an elevated cAMP content. Stimulation of melanogenesis and inhibition of proliferation increased progressively with duration of exposure to theophylline. After 5 days of culture with theophylline, cells were assayed for plating efficiency in theophylline-free medium. Although the number of colony-forming cells was unaffected by previous exposure to theophylline, the colonies were composed of fewer cells inoculated into syngeneic hosts were less tumorigenic than untreated cells. However, theophylline treatment of hosts bearing B16 tumors failed to reduce the tumor growth rate, and theophylline did not potentiate the growth inhibition resulting from treatment with the synthetic polyribonucleotide, polyinosinic-polycytidylic acid.

Animals↗
PubMed1975

Hemolytic anemia induced by murine erythroblastosis virus: possible mechanisms of hemolysis and effects of an interferon inducer.

Murine erythroblastosis virus (MuEV), also called murine leukemia virus-Kirsten, is a member of the murine type-C-RNA leukemia-sarcoma group of oncogenic viruses. Like other members of this group, MuEV can elicit both a hemolytic disorder and an oncogenic response. Neonatal rats infected with MuEV succumb to this hemolytic disorder unless they are treated with the synthetic double-stranded polyribonucleotide, polyinosinic-polycytidylic acid (poly I-poly C). Animals receiving poly I-poly C had markedly reduced levels of virus reproduction as measured by bioassay and electron microscopy. The proliferation of erythroblasts after MuEV infection in animals not receiving poly I-poly C appeared to be an erythropoietin-dependent compensatory response to hemolysis. The hemolysis itself seemed to require virus reproduction in the cell types affected. Administration of poly I-poly C to MuEV-infected rats inhibited virus reproduction and thus may circumvent the hemolytic disease syndrome. The ultrastructure of the virus and of the virus reproduction was also studied.

Anemia, Hemolytic↗
PubMed1974

A simplified method for study of RNA conformation--reaction of formylmethionine transfer RNA with [14C]methylamine-bisulfite.

A new chemical method for radioactive labeling of single-stranded regions of RNA has been used to probe the three-dimensional structure of E. coli tRNA(fMet) in solution. The procedure involves conversion of cytosine residues to N(4)-[(14)C]methylcytosines by treatment with (14)CH(3)NH(2) and sodium bisulfite at pH7. Ribonuclease digestion of the modified tRNA produces (14)C-labeled oligonucleotides which comigrate with the corresponding unlabeled oligonucleotides, facilitating structural analysis. By this procedure, E. coli tRNA(fMet) has been found to contain only six reactive cytosines: C(1), C(16), C(17), C(35), C(75) and C(76). In addition, slow reaction at C(m) (33) was observed. These results are in excellent agreement with previously reported data on the sites of exposed cytosine residues in tRNA(fMet) obtained by two other chemical methods. The methylamine-bisulfite procedure is recommended for studying the ordered structure of more complex polyribonucleotides such as viral and ribosomal RNAs.

Artifacts↗
PubMed1974

Tritium sequence analysis of oligoribonucleotides: a combination of post-labeling and thin-layer chromatographic techniques for the analysis of partial snake venom phosphodiesterase digests.

A tritium derivative method for sequence analysis of polyribonucleotides is detailed, which is based on borotritide reduction of oligonucleotide-3' dialdehydes generated by controlled snake venom phosphodiesterase/alkaline phosphomonoesterase digestion and periodate treatment of time point aliquots of the incubation mixture. Radioactive oligonucleotide derivatives are resolved according to chain length by PEI-cellulose(1) anion-exchange TLC and their 3'-termini identified by techniques described in the preceding paper of this series(2). The present tritium derivative method is compared with the one described previously(2).

Borohydrides↗
PubMed1983

RNA 3'-terminal phosphate cyclase activity and RNA ligation in HeLa cell extract.

HeLa cell extract contains RNA ligase activity that converts linear polyribonucleotides to covalently closed circles. RNA substrates containing 2',3'-cyclic phosphate and 5'-hydroxyl termini are circularized by formation of a normal 3',5' phosphodiester bond. This activity differs from a previously described wheat germ RNA ligase which circularizes molecules with 2',3'-cyclic and 5' phosphate ends by a 2'-phosphomonester, 3',5'-phosphodiester linkage (Konarska et al., Nature 293, 112-116, 1981; Proc. Natl. Acad. Sci. USA 79, 1474-1478, 1982). The HeLa cell ligase can also utilize molecules with 3'-phosphate ends. However, in this case ligation is preceded by an ATP-dependent conversion of the 3'-terminal phosphate to the 2',3' cyclic form by a novel activity, RNA 3'-terminal phosphate cyclase. Both RNA ligase and RNA 3'-terminal phosphate cyclase activities are also present in extract of Xenopus oocyte nuclei, consistent with a role in RNA processing.

Animals↗
PubMed1985

Interaction of DAPI with double-stranded ribonucleic acids.

The interaction of DAPI with natural and synthetic double-stranded polyribonucleotides was studied with different optical and calorimetric methods. The results were similar to those obtained previously with double-stranded polydeoxynucleotides, i.e. two interaction modes, the first of which shows high affinity for AU clusters and consequent strong fluorescence enhancement. The results suggest caution in the use of DAPI as selective fluorescent staining agent for DNA in the presence of RNA. A narrow groove binding model with hydrogen bonds between DAPI and AU pairs is proposed. An intercalation mechanism can be excluded because of the non planarity of DAPI molecule.

Calorimetry↗
PubMed1987

Catalytic activity of the nucleic acid component of the 1,4-alpha-glucan branching enzyme from rabbit muscles.

2.5 S RNA, the nucleic acid component of the 1,4-alpha-D-glucan: 1,4-alpha-D-glucan 6-alpha-(1,4-alpha-glucano)-transferase from rabbit muscles, devoid of any protein, catalyses the branching reaction, as does the holoenzyme. The conclusion is drawn that 2.5 S RNA is a ribozyme. To get an insight into the significance of different parts of the molecule for the catalytic activity of 2.5 S RNA, a large fragment isolated from its partial RNAase A digest was investigated. This fragment which proved to be the middle part of polyribonucleotide chain containing all modified nucleotides exerts some catalytic activity, too.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1988

Evidence for tertiary structure in natural single stranded RNAs in solution.

Binding isotherms (20 degrees C) of ethidium bromide to a number of tRNA species at various ionic strengths indicate that i) the number ni of intercalation sites is high 7 to 11 per molecule, in the low salt form III, but small, 2 to 1, at high Mg2+ or Na+ when form I predominates. ii) modification of tRNA at strategic positions for 3D folding prevents full expression of intercalation restriction iii) maximal restriction is obtained at salt concentrations higher than needed for full conversion to form I. It is inferred that restriction, which is not observed with bihelical RNA (or DNA), requires the native tRNA 3D structure but also some physical coupling between the region of 3D folding and bihelical arms. Ribosomal RNAs, some viral RNAs, mRNA from sheep mammary gland as well as the random copolymers Poly UG, Poly AUG, Poly AUCG all exhibit intercalation restriction. Hence 3D folding of the polyribonucleotide chains appears to be a feature common to single-stranded RNAs when free in solution under physiological conditions.

DNA↗