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PubMed1983

Induction of human leukocyte interferon by heat-treated poly I: poly C.

Synthetic double stranded RNA (poly I: poly C) was prepared from polyinosinic acid (poly I) and polycytidylic acid (poly C) by heat-treated followed by gradual cooling, and was used for induction of human leukocyte interferon (IFN). When poly I: poly C solution was heated at 37 - 65 degrees C for 30 minutes, high activity of human IFN (10,000 - 60,000 i. u./ml) was obtained. In this system, the optimum molecular weight of poly I: poly C to induce IFN was 12.6 - 17.6 S. The properties of induced IFN were heat- and acid- stable, and it was neutralized with anti-IFN alpha serum. So, it was confirmed that it was IFN alpha.

Dose-Response Relationship, Drug↗
PubMed1983

Allosteric effects of cortisol, estradiol, progesterone and of the DNA-sequences poly d(A-T) and poly d(C-G) on the adenosine and thymidine phosphorylating activity of the nuclear nucleoside-nucleotide phosphotransferase C.

The ATP-synthesis of the nuclear nucleoside-nucleotide phosphotransferase C was stimulated by progesterone alone and in combination with poly d(A-T) at 10(-10)-10(-9) M, by estradiol/poly d(A-T) at 5-10(-9) M, by cortisol alone and in combination with poly d(A-T) at 10(-9)-10(-8) M and by poly d(A-T) alone. No effect was observed using poly d(C-G). Using increasing adenosine/dTTP as substrates, cortisol, progesterone, estradiol/poly d(A-T) and poly d(A-T) alone caused positive cooperativity of the substrate saturation curves of the allosteric enzyme C by lowering the apparent Ks 0.5-values and by altering Vmax. The dTTP-synthesis of enzyme C was stimulated by both poly d(A-T) and poly d(C-G) alone and in combination with low estradiol (up to 10(-10) M)-and with higher progesterone concentrations (10(-8)-10(-7) M), whereas cortisol inhibited at higher concentrations completely. Using increasing thymidine/ATP as substrates progesterone and estradiol, also in combination with poly d(A-T) were positive effectors of the substrate saturation curves of enzyme C. By this, the apparent Ks 0.5-values or Vmax were changed. Cortisol could be shown to be a negative effector.

Adenosine↗
PubMed2006

Stability of casein mRNA is ensured by structural interactions between the 3'-untranslated region and poly(A) tail via the HuR and poly(A)-binding protein complex.

The maintenance of mRNA stability has emerged as a mechanism of post-transcriptional control. We demonstrate that beta-casein mRNA stability is dictated by the poly(A) tail and sequences in the 3'-UTR. An in vitro mRNA decay assay revealed that beta-casein mRNA with a long poly(A) tail had higher stability than that with a short poly(A) tail. The addition of poly(A) homopolymer and 3'-UTR cRNA as competitor induced rapid degradation of beta-casein mRNA. The interaction between full-length beta-casein mRNA and poly(A) homopolymer was inhibited by the addition of the 3'-UTR cRNA in the lysates, which indicates that one region of the 3'-UTR associates with the poly(A) tail through an RNA-protein interaction. The putative AU-rich element (ARE) is present at nt 897-905; deletion and mutation analysis showed that the ARE site was required for maintaining the stability of the beta-casein 3'-UTR. In the immunoprecipitation analysis, the poly(A)-binding protein (PABP) and the RNA-binding protein HuR were pulled down by 3'-UTR cRNA, and the absence of the ARE site reduced the binding of these proteins. These experiments further revealed that the rapid degradation of beta-casein mRNA was induced by incubation with HuR- and PABP-depleted RRLs. Collectively, our results suggest that beta-casein mRNA is protected from degradation by virtue of the structural interaction between the 3'-UTR and poly(A) tail via a protein complex of HuR and PABP.

3' Untranslated Regions↗
PubMed1997

Temperature effects on the structure of poly(dA).poly(dT): an X-ray fiber diffraction study.

X-ray fiber diffraction of poly(dA).poly(dT) subjected to variation in the relative humidity, has allowed us to demonstrate the effects of temperature on the conformation of the polynucleotide. When the temperature of the poly(dA).poly(dT) is greater than 30 degrees C and the relative humidity near 80%, a new diffraction pattern is obtained. We observe a transition between the classical alpha B' form of poly(dA).poly(dT) and a double helical structure, B*, which remains stable at a temperature up to 70 degrees C. This new conformation of poly(dA).poly(dT) is a right-handed double helix with 11.4 nucleotide pairs per turn and a pitch of 36.7 A.

Humidity↗
PubMed1979

Cooperative binding of a platinum metallointercalation reagent to poly(A).poly(U).

The cationic complex (2-hydroxyethanethiolato)(2,2',2''-terpyridine)platinum(II), [(terpy)Pt(HET)]+, binds cooperatively to poly(A).poly(U) by intercalation. The melting temperature of poly(A).poly(U) in low-salt buffer is increased by 6 degrees C in the presence of [(terpy)Pt(HET)]+, indicating stabilization of the duplex structure by the bound platinum reagent. Viscosity measurements provide evidence for comparable lengthening of the polynucleotide in the presence of [(terpy)Pt(HET)]+ and the intercalating dye, ethidium bromide. Scatchard plots of the binding of [(terpy)Pt(HET)]+ to poly(A).poly(U) and poly(I).poly(C), determined through ultracentrifugation pelleting methods, show large positive curvature, reflecting the strong cooperativity associated with the platinum complex-RNA interaction. The characteristics of the binding isotherms are interpreted in terms of a model where cooperative pair units of [(terpy)Pt(HET)]+ intercalate into the double-stranded polymer. At saturation, two platinum molecules are bound for every three base pairs. This stoichiometry may be compared with the nearest-neighbor-exclusion binding observed previously in the interaction of [(terpy)Pt(HET)]+ and the ethidium cation with DNA, in which one intercalator occupies every other interbase-pair site at saturation. The striking differences observed in the interaction of [(terpy)Pt(HET)]+ with DNA and RNA suggest that drug recognition is sensitive to the constraints imposed by nucleic acid secondary structure.

Chemical Phenomena↗
PubMed2003

Poly(A)-dependent transcription termination: continued communication of the poly(A) signal with the polymerase is required long after extrusion in vivo.

Genes encoding polyadenylated mRNAs depend on their poly(A) signals for termination of transcription. An unsolved problem is how the poly(A) signal triggers the polymerase to terminate. A popular model is that this occurs during extrusion of the poly(A) signal, at which time it interacts with factors on the transcription complex. To test this idea we used cis-antisense inhibition in vivo to probe the temporal relationship between poly(A) signal extrusion and the commitment of the polymerase to terminate. Our rationale was to inactivate the poly(A) signal at increasing times post-extrusion to determine the point beyond which it is no longer required for termination. We found that communication with the polymerase is not temporally restricted to the time of poly(A) signal extrusion, but is ongoing and perhaps random. Some polymerases terminate almost immediately. Others have yet to receive their termination instructions from the poly(A) signal even 500 bp downstream, as indicated by the ability of an antisense at this distance to block termination. Thus, the poly(A) signal can functionally interact with the polymerase at considerable distances down the template. This is consistent with the emerging picture of a processing apparatus that assembles and matures while riding with the polymerase.

Animals↗
PubMed1985

Netropsin specifically recognizes one of the two conformationally equivalent strands of poly(dA).poly(dT). One dimensional NMR study at 500 MHz involving NOE transfer between netropsin and DNA protons.

Recent observations that the heteronomous structural model for poly(dA).poly(dT) is not found in solution and that in this DNA, the two strands are conformationally equivalent (J. Biomole. Str. Dyns. 2, 1057 (1985], has added a new dimension to the structural dynamics of DNA-netropsin complex. Does the antibiotic somehow distinguish between the two strands and specifically interact with only one of the conformationally equivalent strands? Model-building studies suggest that netropsin can either bind to the dA-strand in the minor groove such that H-bonds are formed between the imino protons N4-H, N6-H, N8-H of netropsin and N3 atoms of A or can bind to the dT-strand in the minor groove and form H-bonds between the imino-protons N4-H, N6-H, N8-H of netropsin and O2 atoms of T. If netropsin binds to the dA-strand, AH2 atoms of poly(dA).poly(dT) would be in closer proximity to the imino protons N4-H, N6-H, N8-H and pyrrole ring protons C5-H, C11-H of netropsin than they would be, if netropsin binds to the dT-strand. In order to distinguish these possibilities experiments were conducted which involved NOE energy transfer between netropsin and DNA protons in the drug-DNA complex. Difference NOE spectra of netropsin-poly(dA).poly(dT) complex in which AH2 was irradiated indicate that dominant NOEs were observed at the imino and pyrrole ring protons of netropsin. When the netropsin pyrrole ring protons were irradiated, the magnetization transfer was at AH2 of DNA. These observations suggest that netropsin binds to the dA-strand of poly(dA).poly(dT) even though dA/dT strands are conformationally equivalent.

Guanidines↗
PubMed1987

The structure of poly(dA).poly(dT) as revealed by an X-ray fibre diffraction.

X-ray diffraction in fibres revealed that the calcium salt of poly(dA).poly(dT) is a 10-fold double helix with a pitch of 3.23 nm. The opposite sugar-phosphate chains in the refined model are characterized by a complete conformational equivalence and contain sugars in a conformation close to C2'-endo. As a result a new model of the sodium salt of poly(dA).poly(dT) has been constructed, which is different from the Heteronomous DNA proposed earlier (S. Arnott et al., Nucl. Acids Res. 11, 4141 (1983)). The new model of Na-poly(dA).poly(dT) has conformationally similar opposite chains; it is a structure of the B-type, rather like that of Ca-poly(dA).poly(dT).

Calcium↗
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↗
PubMed1990

Poly(dA).poly(dT) rich sequences are not sufficient to exclude nucleosome formation in a constitutive yeast promoter.

It was suggested that poly(dA).poly(dT) rich sequences in yeast Saccharomyces cerevisiae act as elements of constitutive promoters by exclusion of nucleosomes (Struhl, K. (1985). Proc. Natl. Acad. Sci. USA 82, 8419-8423). We have mapped the chromatin structure of the pet56-his3-ded1 region in minichromosomes and show that the poly(dA).poly(dT) sequences are located in nuclease sensitive regions. DNA fragments from the nuclease sensitive promoter region of DED1 were used for nucleosome reconstitution in vitro. We show that all sequences can form nucleosome cores and that the poly(dA).poly(dT) sequence can be incorporated in nucleosome cores. The results suggest that the nuclease sensitivity found in vivo is not established by poly(dA).poly(dT) mediated exclusion of nucleosomes.

Base Sequence↗
PubMed1990

CD evidence that the alternating purine-pyrimidine sequence poly[d(A-C).d(G-T)], but not poly[d(A-T).d(A-T)], undergoes an acid-induced transition to a modified secondary conformation.

Circular dichroism and UV absorption data showed that poly[d(A-C).d(G-T)] (at 0.01M Na+ (phosphate), 20 degrees C) underwent two reversible conformational transitions upon lowering of the pH. The first transition was complete at about pH 3.9 and resulted in an acid form of the polymer that was most likely a modified, protonated duplex. The second transition occurred between pH 3.9 and 3.4 and consisted of the denaturation of this protonated duplex to the single strands. UV absorption and CD data also showed that the separated poly[d(A-C)] strand formed two acid-induced self-complexes with pKa values of 6.1 and 4.7 (at 0.01M Na+). However, neither one of these poly[d(A-C)] self-complexes was part of the acid-induced rearrangements of the duplex poly[d(A-C).d(G-T)]. Acid titration of the separated poly[d(G-T)] strand, under similar conditions, did not show the formation of any protonated poly[d(G-T)] self-complexes. In contrast to poly[d(A-C).d(G-T)], poly[d(A-T).d(A-T)] underwent only one acid-induced transition, which consisted of the denaturation of the duplex to the single strands, as the pH was lowered from 7 to 3.

Base Sequence↗
PubMed1990

A porphyrin-DNA exciplex: resonance Raman spectra of electronically excited Cu(TMpy-P4) bound to poly(dA-dT).poly(dA-dT).

The resonance Raman spectra of water-soluble porphyrins, Cu(TMpy-P4) and Ni(TMpy-P4), and their mixtures with DNA, Poly(dG-dC).Poly(dG-dC), and Poly(dA-dT).Poly(dA-dT) were measured using 426 nm pulsed laser excitation (and 556 nm for some applications). At high laser power, the solution of Cu(TMpy-P4) mixed with DNA or Poly(dA-dT).Poly(dA-dT) exhibits new bands at 1550 and 1349 cm-1 that are not observed for Cu(TMpy-P4) alone or for Cu(TMpy-P4) mixed with Poly(dG-dC).Poly(dG-dC). These extra bands do not appear when the resonance Raman spectra are measured by a cw laser or by a pulsed laser with low power. Similar mixtures of M(TMpy-P4) (where M = Ni, Zn, Co, Mn, and H2) with these nucleic acids exhibit no such bands even by high power pulsed laser excitation. We attribute the new resonance Raman bands to an electronically excited Cu(TMpy-P4), stabilized by forming an exciplex with the A-T site of the nucleic acid. The minimum lifetime value of such an exciplex was estimated to be on the order of 10 ps.

Copper↗
PubMed2001

Translational silencing of ceruloplasmin requires the essential elements of mRNA circularization: poly(A) tail, poly(A)-binding protein, and eukaryotic translation initiation factor 4G.

Ceruloplasmin (Cp) is a glycoprotein secreted by the liver and monocytic cells and probably plays roles in inflammation and iron metabolism. We showed previously that gamma interferon (IFN-gamma) induced Cp synthesis by human U937 monocytic cells but that the synthesis was subsequently halted by a transcript-specific translational silencing mechanism involving the binding of a cytosolic factor(s) to the Cp mRNA 3' untranslated region (UTR). To investigate how protein interactions at the Cp 3'-UTR inhibit translation initiation at the distant 5' end, we considered the "closed-loop" model of mRNA translation. In this model, the transcript termini are brought together by interactions of poly(A)-binding protein (PABP) with both the poly(A) tail and initiation factor eIF4G. The effect of these elements on Cp translational control was tested using chimeric reporter transcripts in rabbit reticulocyte lysates. The requirement for poly(A) was shown since the cytosolic inhibitor from IFN-gamma-treated cells minimally inhibited the translation of a luciferase reporter upstream of the Cp 3'-UTR but almost completely blocked the translation of a transcript containing a poly(A) tail. Likewise, a requirement for poly(A) was shown for silencing of endogenous Cp mRNA. We considered the possibility that the cytosolic inhibitor blocked the interaction of PABP with the poly(A) tail or with eIF4G. We found that neither of these interactions were inhibited, as shown by immunoprecipitation of PABP followed by quantitation of the poly(A) tail by reverse transcription-PCR and of eIF4G by immunoblot analysis. We considered the alternate possibility that these interactions were required for translational silencing. When PABP was depleted from the reticulocyte lysate with anti-human PABP antibody, the cytosolic factor did not inhibit translation of the chimeric reporter, thus showing the requirement for PABP. Similarly, in lysates treated with anti-human eIF4G antibody, the cytosolic extract did not inhibit the translation of the chimeric reporter, thereby showing a requirement for eIF4G. These data show that translational silencing of Cp requires interactions of three essential elements of mRNA circularization, poly(A), PABP, and eIF4G. We suggest that Cp mRNA circularization brings the cytosolic Cp 3'-UTR-binding factor into the proximity of the translation initiation site, where it silences translation by an undetermined mechanism. These results suggest that in addition to its important function in increasing the efficiency of translation, transcript circularization may serve as an essential structural determinant for transcript-specific translational control.

3' Untranslated Regions↗
PubMed2000

Localization of poly(A)-binding protein 2 (PABP2) in nuclear speckles is independent of import into the nucleus and requires binding to poly(A) RNA.

The nuclei of mammalian cells contain domains, termed nuclear speckles, which are enriched in splicing factors and poly(A) RNA. Although nuclear speckles are thought to represent reservoirs from which splicing factors are recruited to sites of transcription and splicing, the presence of poly(A) RNA in these structures remains enigmatic. An additional component of the speckles is poly(A) binding protein 2 (PABP2), a protein that binds with high affinity to nascent poly(A) tails, stimulating their extension and controlling their length. In this work we investigated whether PABP2 contributes to the targeting of poly(A) RNA to the speckles. The results show that localization of PABP2 in speckles is independent of import of the protein into the nucleus. Inhibition of transcription or poly(A) synthesis at the end of mitosis does not affect nuclear import of PABP2 but prevents its localization to speckles. Furthermore, PABP2 mutants with decreased ability to bind to poly(A) fail to localize to speckles. Taken together the results show that PABP2 localizes to the nuclear speckles as a consequence of its binding to poly(A) RNA, contrasting to splicing factors which assemble into speckles in the absence of newly synthesized transcripts.

Biological Transport↗
PubMed1999

[Antiviral activity of poly(A)-poly(U) duplexes modified with cis-diammine-dichloroplatinum (II)].

Polyribonucleotide duplex poly(A).poly(U) was modified with cis-diammine dichloroplatinum (II) (cis-DDP). It was shown that the antiinfluenza protective activity of the modified duplex in mice increased with the degree of modification (rb) rising up to 0.2. The effect was different from that for poly(I).poly(C) and poly(G).poly(C). The interferon titers in the murine brain increased in parallel with increasing of the antiviral activity. It was assumed that the structural specificity of the poly(A).poly(U) duplex was responsible for the phenomenon and that cis-DDP interaction with N(7) atoms of the adenine heterocycles blocked the "abnormal" Hoogsteen pairing of adenines with uracils. As a result the antiviral activity increased because of lowering the quantity of the intramolecular defects and increasing the length of the regular double-stranded regions.

Animals↗
PubMed1991

Poly(A)-poly(U) induces circulating colony-stimulating activity resulting from interactions between endogeneous interleukin 6 and serum components.

The immunostimulant poly(A)-poly(U) induces a rapid enhancement of circulating colony-stimulating activity (CSA) in normal mice, culminating 2 h after i.v. injection. A dose of 200 micrograms per mouse is sufficient for a maximal effect. The colonies formed in response to sera from poly(A)-poly(U)-injected mice are mainly granulocytic with few macrophages. These sera are devoid of detectable interleukin 3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF), but contain large amounts of interleukin 6 (IL-6) that are perfectly correlated with circulating CSA levels. Although, in our hands, IL-6 alone induces no colony formation in the standard methylcellulose colony assay, it is nevertheless requisite for this biological activity because 1) monoclonal antibodies against IL-6 strongly diminish colony formation in response to sera from poly(A)-poly(U)-injected mice, and 2) recombinant (r)IL-6 induces colonies when tested in combination with low amounts of normal murine serum. At the concentrations used (0.3%-2.5%), the latter has no or a very slight effect alone. Low amounts of hematopoietic growth factors, that is, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), GM-CSF, or IL-3 that are almost ineffective in the absence of IL-6 can replace normal serum. Taken together, these data suggest that circulating IL-6, induced by i.v. injection of poly(A)-poly(U), promotes colony formation by interacting with serum components that might be identical with hematopoietic growth factors present in normal serum at subliminal concentrations. Finally, the involvement of lipopolysaccharide (LPS) in this phenomenon has been ruled out by the use of the low responder strain of mice (C3H/HeJ) that leads to similar results.

Animals↗
PubMed1979

Properties of unprimed poly(A)-poly(U) synthesis by Caulobacter crescentus RNA polymerase.

Some properties of unprimed poly(A)-poly(U) synthesis by DNA-dependent RNA polymerase from Caulobacter crescentus were examined. The reaction required ATP and UTP as substrates and manganese as a divalent cation. Rifampicin completely inhibited the reaction at a concentration of 1 micron/ml, and the enzyme catalyzed the polymer synthesis well regardless of the presence of GTP, CTP or both. The chain length of the poly(A)-poly(U) synthesized was about one hundred base pairs, as estimated from a sedimentation velocity and the molar ratio of [3H]AMP to [gamma-32P]ATP incorporated into the poly(A)-poly(U). The reaction was dependent on the square of the enzyme concentration and the enzyme dimers formed complexes with poly(A)-poly(U) during the reaction.

Adenosine Monophosphate↗
PubMed1980

Effect of poly(I). poly(C) on the expression of cellular genes.

The effect of poly(I) . poly(C) on DNA and RNA synthesis induced in chicken embryo fibroblasts stimulated by calf serum from a quiescent to a growing state has been investigated. The results show that 1. poly(I) . poly(C) inhibits the induction of DNA synthesis in CEF stimulated by serum. 2. This inhibitory effect is dependent on the time between administration of poly(I) . poly(C) and stimulation, and is manifested at two discrete time intervals, at quiescent (Go) and late presynthetic (Gp) phases. 3. The 4-h incubation of quiescent cells with poly(I) . poly(C) before stimulation with calf serum inhibits and delays the onset of synthetic (S) phase, as manifested by the inhibition and retardation of induced DNA and RNA synthesis and by differences in the sedimentation profiles of total RNA synthesized at middle presynthetic phase of the stimulated cells.

Animals↗