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Results for “RNA Aggregation”

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PubMed1976

The region of tobacco mosaic virus RNA involved in the nucleation of assembly.

The interaction of TMV RNA with the disk aggregate of TMV protein at the initiation of assembly has been studied by using the techniques of RNA sequencing. The 5' end group has been identified, and shown not to be protected in the early stages of assembly from accessibility to nuclease digestion. A population of RNA fragments of average length 250 nucleotides, originating from a unique region of TMV RNA, is encapsidated by limited assembly, and sufficient sequence information is available to identify certain unusual features. The protected region does not contain highly reiterated simple repeating sequences, but may contain more complicated repeats. The length and complexity of the nucleation region may reflect adaptation to the efficient mediation of the conformational change from disk to helix of TMV protein, besides a requirement for binding to the disk, and this may be an important part of the mechanism of specificity in the nucleation of assembly.

Electrophoresis↗

PubMed1977

Assembly of tobacco mosaic virus in vitro: elongation of partially reconstituted RNA.

It was reported that the rate of elongation of partially reconstituted RNA, if prepared without centrifugation, showed dependency upon the state of protein aggregation like that found when free RNA was used as the nucleic acid source [Butler, P. J. G. (1974) J. Mol. Biol. 82, 333-341]. Butler concluded from this result that disks are the kinetically favored protein source for the elongation of the tobacco mosaic virus rod. We show that partially reconstituted RNA prepared without centrifugation always contains a significant amount of free RNA and conclude that the dependency of its elongation rate upon the state of protein aggregation is due to contamination of his partially reconstituted RNA preparation by free RNA. The elongation rate of partially reconstituted RNA, which had been pelleted and contained no free RNA, was found to be about the same regardless of the state of aggregation of the added protein.

Centrifugation, Density Gradient↗

PubMed1978

Estrogen withdrawal in chick oviduct. Characterization of RNA synthesized in isolated nuclei using a mercurated precursor.

1. As a prerequisite for analyzing the effect of estrogen on transcription in chick oviduct, we describe suitable methods for the synthesis (under conditions restricting reinitiation), and isolation of RNA transcripts from oviduct nuclei in vitro, utilizing mercurated UTP (Hg-UTP) as an RNA precursor and chromatography on sulphydryl-Sepharose (SH-Sepharose) to recover mercurated RNA (Hg-RNA). The techniques described include treatment of Hg-RNA with p-hydroxymercuribenzoate, to improve the efficiency of binding to SH-Sepharose, and elution of Hg-RNA from SH-Sepharose after treatment with 60% formamide at 90 degrees C, to eliminate contamination by aggregated nucleic acid. 2. RNA synthesized by endogenous form B RNA polymerase (using either UTP or Hg-UTP as precursor) was recovered in nuclear lysates in the form of 30--85-S heterogeneous RNA . protein complexes, and after removal of protein, was 10--12 S in size. 3. The nature of RNA transcripts synthesized in vitro was examined by hybridization. More than 90% of the RNA was complementary to "unique" DNA sequences, and 50--60% of the hybridized RNA could be competed with homologous, steady-state nuclear RNA, indicating a significant degree with homologous, steady-state nuclear RNA, indicating a significant degree of homology between in vitro transcripts and in vivo RNA. The level of homology was similar whether RNA synthesis was performed in low salt, or in high salt in the presence of heparin. Possible reasons for only partial competition in these experiments are discussed. 4. Withdrawal of estrogen from chicks leads to a 50% reduction in endogenous RNA polymerase activities in nuclei within 48 h. Similar levels of competition with Hg-RNA transcripts for "unique" DNA were obtained using oviduct nuclear RNAs isolated before or after estrogen withdrawal, and even with liver nuclear RNA. Thus, in oviduct, those sequences present in primary transcripts, and analyzed under our experimental conditions, are present in different hormonal states and also in other chick tissues.

Amanitins↗

PubMed1991

Scanning tunnelling microscopy of 16S ribosomal RNA in water.

The scanning tunnelling microscope has been used to image 16S ribosomal RNA molecules in water electrophoretically deposited on graphite surface. Two kinds of images have been obtained: images showing aggregates of 16S ribosomal RNA molecules similar to those obtained from DNA solutions and others showing individual 16S ribosomal RNA molecules. An interesting characteristic of these images, recorded in constant current mode, is that the 16S ribosomal RNA molecules appear to be located below the graphite surface. The morphology and several structural parameters of the molecules were consistent with the data obtained from electron microscopy.

Centrifugation, Zonal↗

PubMed1990

Switching in the self-assembly of tobacco mosaic virus.

Experimental observations on the structure and physicochemical properties of TMV protein assemblies have led to a fundamental switch in the model of the self-assembly process: rather than being nucleated by the hypothetical two-layer disk, virus assembly appears to be initiated by interaction of the specific RNA sequence with a short helical aggregate of the coat protein arranged as in the virus. Formation of the 20s nucleating aggregate involves the binding of an average of half a proton per protein subunit. This proton-binding site can be identified with the carboxyl-carboxylate pair that is formed between top and bottom protein surfaces at a radius of 58 A in the virus helix. Because the 20s aggregate consists of about two helical turns, only one carboxyl-carboxylate pair will be formed between each top-bottom pair of protein subunits. Limitation of the length of the 20s helical aggregate at neutral pH can be accounted for by disorder of the inner loop of the protein chain, due to electrostatic repulsion among the carboxyl groups that form the anomalous proton-binding site at 25 A radius in the ordered virus structure. To grow beyond two to three turns, inner loops of the protein at the interior of the helix must be ordered in the close-packed arrangement. The electrostatic repulsion opposing this ordering can be overcome by binding of the viral RNA at neutral pH, by calcium binding, or by proton binding in slightly acid solution. Virus disassembly upon infection appears to result from the low intracellular calcium and proton concentration compared to the extracellular environment, which increases the electrostatic repulsion among the negatively charged groups involved in calcium and proton binding, thereby allowing cellular ribosomes to competitively bind the viral RNA. Disk aggregates of TMV protein, which form at high ionic strength in alkaline solution, do not appear to be involved in virus assembly. The stacked-disc aggregate, which was previously presumed to be built of a polar stack of the hypothetical polar two-layer aggregate, is, in fact, a bipolar structure. Because the bonding between turns of the disc structures is different from that of the virus helix, direct switching between these structures by the postulated dislocation does not occur. TMV assembly does appear to involve conservation of bonding specificity, as initially presumed, but only in helical packing arrangements of the protein subunits. Switching from disordered to ordered conformations of the protein, dependent on changes in the electrostatic interactions among the protein subunits, appears to be critical in controlling the assembly process.

Microscopy, Electron↗

PubMed2026

Riboregulation: a non-canonical tau function.

Almost since its discovery, tau protein has perplexed scientists and clinicians with its varied roles in physiology as well as its appearance as phosphorylated protein aggregates of various structures in many neurodegenerative diseases. Tau plays a role in microtubule stabilization, but from the earliest of studies, tau has also been observed to bind to RNA, with recent research suggesting tau has a higher affinity for some RNA species compared to microtubules. In the context of disease, tau dysfunction potentiates disruptions to RNA metabolism, including the perturbation of mRNA splicing, impairment of translation, de-repression of transposable elements, and alteration of RNA export and degradation. Tau aggregates directly sequester diverse RNA species and RNA binding proteins. Emerging evidence reinforces the characterization of tau as an RNA binding protein, highlighting questions about both the physiological and disease-related functions of this direct RNA binding. The disparate structure of tau in normal and various disease states makes teasing apart the various impacts on RNA and regulation a more difficult puzzle requiring future study. In this review, we summarize the evidence for tau's role in RNA biology, including as an RNA binding protein.

tau Proteins↗

PubMed1975

Murine oncornavirus high-molecular-weight RNA structure thermal stepwise dissociation of 70S murine leukemia-sarcoma virus to subunits and low-molecular-weight associated RNAs.

The thermal dissociation into subunits and low-molecular-weight (LMW) associated RNAs of the aggregate structure of 70S RNA of a murine leukemia sarcoma viral complex was studied. By polyacrylamide-agarose gel electrophoresis, it was found that at low temperature a fraction of the genome was converted into an intermediate population of RNA (Im.P) with an apparent molecular weight of 6.6 times 10-6. At higher temperature, the 70S RNA and the Im.P RNA were successively dissociated into two RNA subunits called "I" and "II" and 70S-associated LMW RNAs. The apparent molecular weight of subunit I was about 5 times 10-6 and that of subunit II was about 3.2 times 10-6. The release of 4S, 5S, 5.5S, and 8S RNAs from 70S RNA at various temperatures was studied by composite polyacrylamide gel electrophoresis. It was found that the nature of hydrogen bonding to the 70S RNA was different for each LMW RNA species. A possible relationship of the association between the subunits and each 70S-associated LMW RNA, based on their T-m values, is discussed.

Electrophoresis, Polyacrylamide Gel↗

PubMed1978

Effect of RNase III on the size of bacteriophage T7 lysozyme mRNA.

The size of lysozyme mRNA from T7-infected E. coli RNase III+ and RNase III- strains was analyzed by sucrose gradient sedimentation, dimethylsulfoxide (Me2SO) sucorse gradient sedimentation, and preparative gel electrophoresis. Each technique revealed a similar size distribution of multiple lysozyme mRNA's. Analysis by preparative gel electrophoresis of RNA extracted after infection of Escherichia coli Bst (RNase III+) separated lysozyme mRNA into six peaks of activity ranging in size from 0.2 x 10(6) to 1.9 x 10(6) daltons. Four well-resolved major peaks of activity were detected, having apparent molecular weights of approximately 0.61 x 10(6), 0.76 x 10(6), 0.92 x 10(6), and 1.3 x 10(6). A broad band of activity, with a molecular weight range from 0.2 x 10(6) to 0.37 x 10(6), was also present, and a sixth peak of activity was sometimes observed that migrates with a mobility corresponding to a molecular weight of 1.9 x 10(6). Judging from their molecular weight as estimated by electrophoresis, most, if not all, of the lysozyme mRNA's were polycistronic. The RNA extracted after infection of an RNase III- host contained a more heterogeneous collection of lysozyme mRNA's. In addition to lysozyme mRNA activity on RNAs with molecular weights between 0.2 x 10(6) and 1.9 x 10(6), RNA species with molecular weights estimated at 4 x 10(6) to 5 x 10(6) were also detected. The data indicate that RNase III processes at least some of the primary lysozyme transcripts. The multiple lysozyme mRNA's represent discrete RNA species rather than aggregates because analysis of the size of lysozyme mRNA under completely denaturing conditions, in Me2SO, produced a similar size distribution of lysozyme mRNAs. Also, treatment of RNA with 90% Me2SO, which separates the strands of a completely double-stranded RNA, did not significantly alter the electrophoretic mobility of the lysozyme mRNA.

Coliphages↗

PubMed1977

Purification of a putative precursor of globin messenger RNA from mouse nucleated erythroid cells.

Nucleated erythroid cells were incubated for 10 min in the presence of [5-3H]uridine, and the total RNA was isolated by three different extraction procedures. RNA containing globin messenger RNA sequences was purified from other cellular RNAs by selective hybridization to globin complementary DNA cellulose. Depending upon the extraction procedure employed, 0.4-0.6% of the radioactively-labeled total cellular RNA applied to the column annealed to globin complementary DNA cellulose. The annealed RNA was treated with formaldehyde and analyzed by formaldehyde/polyacrylamide gel electrophoresis. Mature globin mRNA and an RNA migrating at approximately 15 S were observed. No globin mRNA containing sequences larger than 20 S were present. The 15S RNA was partially resolved from mature globin mRNA by neutral sucrose density gradient centrifugation. The RNA isolated from the heavy region of this gradient migrated as 15 S in the formaldehyde/polyacrylamide gels and retained its ability to quantitatively anneal to globin complementary DNA cellulose. On the basis of these observations, we conclude that nucleated erythroid cells obtained from the spleens of anemic mice have a 15S RNA which contains globin mRNA sequences. The 15S RNA is not an aggregate and is a good candidate for a globin mRNA precursor.

Animals↗

PubMed1979

Modulation of platelet activation by native DNA.

Native DNA (dsDNA) was found to induce the aggregation of isolated human platelets and the release of platelet 5HT; this activation was inhibited by both theophylline and TYA, suggesting a role for cAMP and metabolic products formed from arachidonate. By contrast, nonaggregating amounts of dsDNA inhibited platelet activation induced by collagen or thrombin. This inhibition, which could be overcome by use of greater amounts of the stimulatory agents, was not associated with the loss of platelet viability. Activation of platelets by dsDNA was not observed in plasma or in isolated platelet systems to which small amounts of cell-free plasma were added. However, dsDNA maintained in plasma its ability to inhibit platelet aggregation induced by collagen and thrombin. RNA and single-stranded DNA failed to induce platelet aggregation or release of 5HT and to block the platelet activation stimulated by dsDNA. Further, dsDNA did not significantly inhibit platelet aggregation in platelet-rich plasma stimulated by ADP or epinephrine. These data implicate dsDNA as a selective and potentially important activator and modulator of platelet responsiveness.

Adenosine Diphosphate↗

PubMed1978

Role of intracisternal A-particles and of RNA molecules in plasmacytoma-associated immunodeficiency.

A subcellular fraction from murine plasmacytoma cells was shown to suppress the primary antibody response when injected into normal mice. The active subcellular fraction copurified with intracisternal A-particles. The RNA extracted from subcellular fractions enriched in A-particles was also immunosuppressive. This activity was due to a population of RNA molecules that contained polyadenylic acid. Upon fractionation on a sucrose gradient, two populations of immunosuppressive RNA were obtained with sedimentation velocities of 12 to 18S and 40 to 50S. The 40 to 50S RNA was shown to be a thermolabile aggregate of molecules that contained the 12 to 18S RNA molecules. Plasmacytoma-derived material with similar physicochemical characteristics had previously been shown to induce in normal mouse lymphocytes surface immunoglobulins with the plasmacytoma idiotype, supporting the possibility that one of the mechanisms responsible for the development of immunological deficiency is the change of surface immunoglobulins of nonmalignant B-cells.

Animals↗

PubMed1990

The postsynaptic 43K protein clusters muscle nicotinic acetylcholine receptors in Xenopus oocytes.

Nicotinic acetylcholine receptors (AChRs) are localized at high concentrations in the postsynaptic membrane of the neuromuscular junction. A peripheral membrane protein of Mr 43,000 (43K protein) is closely associated with AChRs and has been proposed to anchor receptors at postsynaptic sites. We have used the Xenopus oocyte expression system to test the idea that the 43K protein clusters AChRs. Mouse muscle AChRs expressed in oocytes after injection of RNA encoding receptor subunits are uniformly distributed in the surface membrane. Coinjection of AChR RNA and RNA encoding the mouse muscle 43K protein causes AChRs to form clusters of 0.5-1.5 microns diameter. AChR clustering is not a consequence of increased receptor expression in the surface membrane or nonspecific clustering of all membrane proteins. The 43K protein is colocalized with AChRs in clusters when the two proteins are expressed together and forms clusters of similar size even in the absence of AChRs. These results provide direct evidence that the 43K protein causes clustering of AChRs and suggest that regulation of 43K protein clustering may be a key step in neuromuscular synaptogenesis.

Animals↗

PubMed1992

Mechanisms of fibrin formation and lysis by human lung fibroblasts: influence of TGF-beta and TNF-alpha.

Fibrin gels form within the alveolar and interstitial compartments of the injured lung, and fibroblasts invade and facilitate organization of these transitional gels. We studied the effects of transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) on fibrinolytic and procoagulant activities of human lung fibroblasts (HLF) to determine their capacity to regulate pulmonary fibrin deposition. Fibrinolytic activity of cell lysates and media (n = 6 HLF cultures) were uniformly depressed by TGF-beta or TNF-alpha. In dose and time-course studies, HLF plasminogen activator inhibitor-1 (PAI-1) was increased by TGF-beta, whereas TNF-alpha induced release of PAI-1 into the media. HLF and media urokinase concentrations were depressed by TGF-beta, whereas urokinase was unchanged or increased by TNF-alpha. Tissue plasminogen activator was mainly cell associated and unchanged by TGF-beta or TNF-alpha. HLF antiplasmin activity was not detected. Plasma recalcification times of HLF media were decreased by TNF-alpha but unchanged by TGF-beta. These studies suggest that TGF-beta and TNF-alpha impair the ability of HLF to degrade fibrin by disturbing the balance of HLF plasminogen activators and PAI and that these cytokines concurrently leave unchanged or increase the capacity of HLF to initiate fibrin formation. Cytokines likely to occur in the injured lung induce abnormalities of fibrinolysis in HLF from adults; such abnormalities favor extravascular fibrin deposition, a characteristic feature of alveolitis.

Base Sequence↗

PubMed1992

Protein-protein interactions in high molecular weight forms of the transformation-related phosphoprotein p53.

The transformation-related cellular phosphoprotein p53 interacts with a variety of viral and cellular proteins and with itself to form high molecular weight complexes. The formation of high molecular weight complexes correlates with the transformed morphology of the cells whereas in non-transformed cells low molecular weight forms are predominant. Thus, aggregation seems to be involved in the regulation of biological functions of p53. Analyzing wild-type and mutant p53 in the same cellular environment i.e. after an in vitro transcription/translation reaction in rabbit reticulocytes we found high molecular weight forms for wild-type and mutant p53. The sedimentation profile resembled the profile obtained for mutant p53 from transformed cells. As shown by dilution experiments, aggregation of p53 was not due to high p53 protein concentrations. Although p53 is known to bind RNA, treatment with RNAse did not change the aggregation state of p53 suggesting that RNA may not contribute to the quaternary structure of p53. High molecular weight aggregates of p53 were resistant to treatment with 1 M NaCl and also stable in weak acidic conditions. Alkaline pH as well as treatment with 3.5 M NaCl led to a disaggregation of high molecular weight complexes of p53. This treatment resulted in low molecular weight forms consisting probably of dimers to tetramers whereas monomers of p53 are hardly detectable. A nearly complete disaggregation was obtained only with the ionic denaturing detergent sodium dodecyl sulfate. Therefore, one has to assume different types of protein-protein interactions leading to the various quaternary structures of p53.

Animals↗

PubMed1979

Inhibition of desmosome formation in chick cell aggregates.

Desmosomes (macula adherens) have been associated with the function of adhesion. Their possible role in aggregation and sorting of chick and mouse epithelial cells has been investigated. Treatment of aggregates with 2-5 microgram/ml of actinomycin D which inhibited RNA synthesis also inhibited both desmosome formation and aggregation if administered at the beginning of the aggregation process. In contrast, if the drug was administered at six hours, when the cells had recovered from the process of dissociation, then aggregation over the following six hours appeared normal from observation of living samples. Such aggregates incorporated leucine-3H at roughly 85% of the control level. A quantitative comparison was made of desmosome formation in aggregates treated with actinomycin D for hours 6-12 and those cultured in normal medium. Desmosome formation was inhibited by the drug, although aggregation could proceed. Combinations of chick corneal and mouse skin cells sorted out in the presence of actinomycin D to the same extent as controls. Thus desmosome formation, which normally occurs during aggregation of the epithelial cells studied here, is not coupled with the aggregation or cell sorting process in these cells of stratified epithelia. When cells were treated with cycloheximide (100 muM) both desmosome formation and the progressive rounding up of aggregates was inhibited.

Animals↗

PubMed1977

Molecular changes in lymph node tissue after skin heterografts.

Changes in the rat lymph node RNA base composition and in ribosomal aggregation induced by heterologous skin graft have been characterized by the base composition analysis of the phenol extractable RNA, by the density gradient centrifugation of the ribosomal particles and by amino acid incorporation in the newly synthetized protein. Following skin grafts, the changes in base composition of total RNA in lymph . nodes were mainly due to the synthesis of ribosomal type RNA. Ribosomes were assembled as single units or as polyribosomes. The present findings also show an increase in amino acid incorporation by polyribosomes after the first graft reaction.

Amino Acids↗

PubMed1979

The identification of globin messenger ribonucleic acid in newt erythropoietic cells.

Polyadenylated [poly(A)+]-RNA isolated from newt (Triturus cristatus) erythropoietic cells contained two main species sedimenting at 9S and 25S, and minor amounts of a 15-20S component. The 9S poly(A)+-RNA fraction induced synthesis of newt haemoglobin and globins in frog oocytes and in an mRNA-dependent rabbit reticulocyte lysate, confirming its identity as newt globin mRNA. Translation of 9S globin mRNA in reticulocyte lysate was concentration-dependent, the patterns of globin synthesis suggesting both preferential utilization and unequal amounts of the different globin mRNA subspecies. Globin mRNA activity was also evident in the 25S poly(A)+-RNA fraction whose localization in polyribosomes excluded its function as a nuclear globin mRNA precursor. Denaturation in formamide and estimation of its relative methyl content indicated that the 25S poly(A)+-RNA fraction contained equimolar amounts of 9S globin mRNA and 26S rRNA. Translation of the 25S fraction in reticulocyte lysate was less efficient than that of comparable amounts of 9S globin mRNA and induced a pattern of globin synthesis similar to that obtained with subsaturating amounts of 9S mRNA. The 25S mRNA-rRNA complex was considered to be a non-physiological aggregate generated by extraction of RNA in the presence of buffers of moderate to high ionic strength.

Animals↗

PubMed1979

Ribosomal ribonucleic acid isolated from Salmonella typhimurium: absence of the intact 23S species.

Ribonucleic acid (RNA) isolated by four distinct methods and from a variety of Salmonella typhimurium strains lacked intact 23S ribosomal RNA (rRNA). On sucrose gradients which minimize aggregation, the vast majority of S. typhimurium rRNA sedimented as a 16S peak with a 14S shoulder. RNA from this region of the gradient was resolved into three discrete bands by electrophoresis in formamide. Two very minor S. typhimurium RNA peaks were resolved at 21S and 10S on sucrose gradients, and each peak formed discrete bands in electrophoresis. It is concluded that if S. typhimurium does possess an intact 23S rRNA species, this species is extremely "labile." The absence of isolatable S. typhimurium 23S rRNA possibly reflected in vivo processing of the rRNA before isolation. Under certain conditions, S. typhimurium rRNA formed discrete aggregates which sedimented similarly to intact Escherichia coli 23S rRNA.

Centrifugation, Density Gradient↗