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At least 235 records · Page 13Linked to original sources
PubMed1995

Identification of the cauliflower mosaic virus movement protein RNA-binding domain.

The in vitro RNA-binding activity of the movement protein (P1) of cauliflower mosaic virus was studied after its expression in Escherichia coli, purification, and uv-crosslinking to a radioactive probe. It was found that insoluble P1 aggregates were involved in RNA-binding activity. A series of deletion mutants were used to identify a domain within P1 required for binding activity. The RNA-binding domain is located between amino acids 120 and 197 and includes the region of homology between P1 and the movement protein (P30) of tobacco mosaic virus (TMV). The homologous region corresponds to part of RNA-binding domain "A" in TMV P30, but unlike domain A, the P1 domain is able to bind RNA out of the context of the complete protein. The P1 RNA-binding domain shows some structural similarity with RNA-binding domains of other proteins. The conservation of this domain in the caulimo- and badnaviruses provides support for the view that this activity has biological relevance.

Amino Acid Sequence↗
PubMed1989

Vesicular stomatitis virus RNA replication: a role for the NS protein.

Synthesis of the vesicular stomatitis virus nucleocapsid (N) protein is required for viral RNA replication. The observation that the N protein forms a rapidly sedimenting species in the absence of other viral proteins and the description of complexes of N protein with NS protein led to the proposal that NS protein binds to N protein to prevent it from self-associating. We tested this model by analysing the physical properties of N protein synthesized alone in an in vitro replication system as compared to N protein synthesized in the presence of the NS protein. These findings were correlated with the ability of the N protein, synthesized under both conditions, to support replication. N protein synthesized at low concentrations in the absence of other viral proteins sedimented at 4S on glycerol gradients and was capable of supporting RNA replication. In contrast, synthesis of increasing concentrations of N protein resulted in formation of a rapidly sedimenting species of N protein which had the physical properties of a protein-protein aggregate and which failed to support RNA replication. Co-synthesis of the NS protein with N protein both prevented the concentration-dependent aggregation of N and restored the ability of high concentrations of N protein to support RNA replication.

Capsid↗
PubMed2006

Monitoring granule formation in anaerobic upflow bioreactors using oligonucleotide hybridization probes.

The process of granule formation in upflow anaerobic sludge blanket (UASB) reactors was studied using oligonucleotide hybridization probes. Two laboratory-scale UASB reactors were inoculated with sieved primary anaerobic digester sludge from a municipal wastewater treatment plant and operated similarly except that reactor G was fed glucose, while reactor GP was fed glucose and propionate. Size measurements of cell aggregates and quantification of different populations of methanogens with membrane hybridization targeting the small-subunit ribosomal RNA demonstrated that the increase in aggregate size was associated with an increase in the abundance of Methanosaeta concilii in both reactors. In addition, fluorescence in situ hybridization showed that the major cell components of small aggregates collected during the early stages of reactor startup were M. concilii cells. These results indicate that M. concilii filaments act as nuclei for granular development. The increase in aggregate size was greater in reactor GP than in reactor G during the early stages of startup, suggesting that the presence of propionate-oxidizing syntrophic consortia assisted the formation of granules. The mature granules formed in both reactors exhibited a layered structure with M. concilii dominant in the core, syntrophic consortia adjacent to the core, and filamentous bacteria in the surface layer. The excess of filamentous bacteria caused delay of granulation, which was corrected by increasing shear through an increase of the recycling rate.

Bioreactors↗
PubMed1998

Nuclear RNA is extruded from apoptotic cells.

During spontaneous apoptosis of thymocytes there is extrusion of ribonucleoproteins (RNPs) from the cell. The aim of this investigation was to elucidate whether the RNP aggregates in apoptotic cells and bodies still contain RNA in an appreciable amount. We demonstrated by specific cytochemical techniques that the aggregates of nuclear RNPs extruded in the cytoplasm of spontaneously apoptotic thymocytes contain RNA in a sufficient amount to be detected cytochemically. These heterogeneous ectopic RNP-derived structures (HERDS) are formed by perichromatin fibrils, interchromatin granules, perichromatin granules, and nucleolar material. The RNA detected inside these clusters should therefore correspond to both mRNA and snRNA as well as to rRNA. We never observed DNA-containing aggregates in the cytoplasm of apoptotic thymocytes. The presence of RNA in the HERDS that may be released from apoptotic cells suggests that the decrease in the amount of total RNA during apoptosis may be mostly linked to cellular extrusion rather than to degradation of RNA by RNase activities. Another interesting aspect of these results lies in the hypothesis of apoptosis as a possible cause for the presence of autoantibodies in the serum of patients with systemic autoimmune diseases.

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↗
PubMed2003

Adenine and RNA in mineral samples. Surface-enhanced Raman spectroscopy (SERS) for picomole detections.

Studies on the interactions of biological macromolecules with mineral surfaces are crucial for the detecting biomarkers. But before this can be done for real samples like rocks or sediments, rational methods based on mineral models plus known amounts of nucleic acids must be developed. The methods must be very sensitive, as the amount of bound macromolecule may be very small. Surface-enhanced Raman spectroscopy (SERS) is perfect for detecting picomolar amounts of nucleic acid materials. In this study, the models used were adenine and GAAA hairpin for nucleic acids materials and a clay (montmorillonite) plus colloidal silver (used for SERS detection) for mineral supports. We have shown that OH(-) anions compete with adenine and the adenyl residues in the GAAA loop for adsorption onto nano-sized silver particles in basic medium. The GAAA adenyl moieties are less well adsorbed onto either clay or silver than is adenine. Also, the transfer of either adenine or the RNA hairpin from the clay to the silver aggregates is pH-dependent. Contact between adenine and the montmorillonite also seems to disperse adenine aggregates. The clay could also increase the flexibility of the RNA hairpin so that it is released from the clay at pH 10, and the affinity of its adenyl moieties for the metallic substrate is enhanced.

Adenine↗
PubMed1971

Transcription of the polyoma virus genome: synthesis and cleavage of giant late polyoma-specific RNA.

The size of virus-specific RNA synthesized in cultured mouse kidney cells infected with polyoma virus was estimated by electrophoresis and sedimentation analysis of RNA extracts from whole cells. Newly synthesized "late" polyoma-specific RNA appears as "giant" molecules of heterogeneous size, up to several times larger than a strand of polyoma DNA (1.5 x 10(6) daltons). Treatment with dimethylsulfoxide or urea showed that the large size of these molecules is not due to aggregation. Giant polyoma-specific RNA is strikingly similar in size distribution to "nuclear messenger-like" RNA ("heterogeneous nuclear" RNA) of the host cell. Subsequent to its synthesis, some of the giant polyoma-specific RNA appears to be cleaved to at least three smaller species.

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↗
PubMed1987

Isolation and characterization of nucleoprotein assembly intermediates of tobacco mosaic virus.

During assembly of tobacco mosaic virus from pure RNA and 20S capsid protein aggregates under conditions where protein is limiting, partially assembled intermediates of specific sizes accumulate; these were isolated on sucrose density gradients. The earliest intermediate found in substantial quantity sedimented at 56 S and was shown, by measurement of its buoyant density and of the length of the RNA segment protected by the capsid protein from nuclease digestion, to consist of RNA that is 13% encapsidated (corresponding to a rod length of about 39 nm); the next intermediate sediments at 78 S and is 18% encapsidated (corresponding to a rod length of about 54 nm). Studies of the distribution of intermediates at various input ratios of protein/RNA indicated that their accumulation results from decreases in the rate constants for protein binding that are local to specific points in the course of encapsidation. After extensive nuclease digestion, the RNA still associated with the first intermediate was shown to include a portion that is unencapsidated. This segment of the RNA may be a region of stable secondary that confers the nuclease resistance despite the lack of protection by capsid protein. Such RNA secondary structure, if it exists, would also cause the accumulation of intermediates by imposing an energy barrier to subsequent rod elongation.

Capsid↗
PubMed1996

Differential compartmentalization of vasopressin messenger RNA and neuropeptide within the rat hypothalamo-neurohypophysial axonal tracts: light and electron microscopic evidence.

Arginine vasopressin messenger RNA is axonally transported in the rat hypothalamo-neurohypophysial system [for review see Mohr et al. (1993) In Vasopressin (eds Gross P., Richter D. and Robertson C. L.), pp. 119-129, John Libbey Eurotext]. Upon chronic dehydration (2% saline-feeding for seven days), vasopressin messenger RNA within this axonal compartment is dramatically increased and appears aggregated in a selected subset of axonal swellings confined to the median eminence and posterior pituitary. In this study, we analysed the axonal distribution of the vasopressin messenger RNA within the hypothalamo-neurohypophysial tracts of control and saline-fed animals, and compared this distribution to that of the vasopressin peptide. Our data further support a selective aggregation of the vasopressin messenger RNA in a subset of distal axonal swellings and/or terminals of the median eminence and posterior pituitary. The selective aggregation is observed not only in saline-fed animals, but also in control animals. Although the osmotic stimulus dramatically enhances the axonal transport of vasopressin messenger RNA, the consequent general distribution pattern of the messenger RNA in the hypothalamo-neurohypophysial system is not changed. However, the physiological perturbation does increase the number of vasopressin messenger RNA-containing swellings within the median eminence and the posterior pituitary. In both saline-fed and control animals, the level of messenger RNA label within individual swellings appeared roughly similar to that found in the perikaryal cytoplasm of extra-hypothalamic vasopressinergic neurons. A detailed comparison of the axonal compartmentalization of vasopressin messenger RNA and vasopressin peptide demonstrates that the axonal distribution of vasopressin messenger RNA does not precisely overlap that of vasopressin peptide along the hypothalamo-neurohypophysial tract. In seven-day saline-fed animals, the majority of the messenger RNA-containing swellings of the median eminence also contain detectable vasopressin peptide; however in the same animals, nearly all the messenger RNA-containing swellings of the posterior pituitary appear devoid of vasopressin peptide. Therefore, our work strongly suggests that at least in the posterior pituitary, the vasopressin messenger RNA might be selectively targeted and aggregated in a selected subset of axonal swellings containing little if any vasopressin, and hence very few neurosecretory granules. Given this evidence that vasopressin messenger RNA and neuropeptide are differentially compartmentalized in axons of magnocellular neurons, we propose that vasopressin messenger RNA and peptide probably rely on different intracellular transport systems with respect to packaging, transport and/or aggregation within these selected axonal locations.

Animals↗
PubMed2006

Transmission electron microscopy reveals an optimal HIV-1 nucleocapsid aggregation with single-stranded nucleic acids and the mature HIV-1 nucleocapsid protein.

HIV-1 nucleocapsid protein (NCp7) condenses the viral RNA within the mature capsid. In a capsid-free system, NCp7 promotes an efficient mechanism of aggregation with both RNA and DNA. Here, we show an analysis of these macromolecular complexes by dark-field imaging using transmission electron microscopy. Thousands of mature NCp7 proteins co-aggregate with hundreds of single-stranded circular DNA molecules (ssDNA) within minutes, as observed with poly(rA). These co-aggregates are highly stable but dynamic structures, as they dissociate under harsh conditions, and after addition of potent ssDNA or NCp7 competitive ligands. The N-terminal domain and zinc fingers of NCp7 are both required for efficient association. Addition of magnesium slightly increases the avidity of NCp7 for ssDNA, while it strongly inhibits co-aggregation with relaxed circular double-stranded DNA (dsDNA). This DNA selectivity is restricted to mature NCp7, compared to its precursors NCp15 and NCp9. Moreover, for NCp15, the linkage of NCp7 with the Gag C-terminal p6-peptide provokes a deficiency in ssDNA aggregation, but results in DNA spreading similar to prototypical SSB proteins. Finally, this co-aggregation is discussed in a dynamic architectural context with regard to the mature HIV-1 nucleocapsid. On the basis of the present data, we propose that condensation of encapsidated RNA requires the C-terminal processing of NCp. Subsequently, disassembly of the nucleocapsid should be favoured once dsDNA is produced by HIV-1 reverse transcriptase.

Capsid Proteins↗
PubMed2000

Dystroglycan overexpression in vivo alters acetylcholine receptor aggregation at the neuromuscular junction.

Dystroglycan is a member of the transmembrane dystrophin glycoprotein complex in muscle that binds to the synapse-organizing molecule agrin. Dystroglycan binding and AChR aggregation are mediated by two separate domains of agrin. To test whether dystroglycan plays a role in receptor aggregation at the neuromuscular junction, we overexpressed it by injecting rabbit dystroglycan RNA into one- or two-celled Xenopus embryos. We measured AChR aggregation in myotomes by labeling them with rhodamine-alpha-bungarotoxin followed by confocal microscopy and image analysis. Dystroglycan overexpression decreased AChR aggregation at the neuromuscular junction. This result is consistent with dystroglycan competition for agrin without signaling AChR aggregation. It also supports the hypothesis that dystroglycan is not the myotube-associated specificity component, (MASC) a putative coreceptor needed for agrin to activate muscle-specific kinase (MuSK) and signal AChR aggregation. Dystroglycan was distributed along the surface of muscle membranes, but was concentrated at the ends of myotomes, where AChRs normally aggregate at synapses. Overexpressed dystroglycan altered AChR aggregation in a rostral-caudal gradient, consistent with the sequential development of neuromuscular synapses along the embryo. Increasing concentrations of dystroglycan RNA did not further decrease AChR aggregation, but decreased embryo survival. Development often stopped during gastrulation, suggesting an essential, nonsynaptic role of dystroglycan during this early period of development.

Animals↗
PubMed1980

RNA-protein interactions in the assembly of tobacco mosaic virus.

Assembly of tobacco mosaic virus is initiated by the binding of a specific loop of the RNA into the central hole of the disk aggregate of protein subunits. Since the nucleation loop is located about five-sixths along the RNA molecule, subsequent elongation must be bidirectional. We have now measured the rates of elongation in the two directions by determining the lengths of RNA protected from nuclease digestion at different times and using either intact TMV rNA, or RNA with most of the longer tail removed. Comparison of the rates with the protein supplied as either a mixture of disks with A-protein (a mixture of less aggregated states) or just A-protein, shows that different mechanisms and protein aggregates are used for the most rapid growth. When disks are present, they add more rapidly along the longer RNA tail but do not appear to add directly on the shorter tail. In contrast, smaller aggregates (A-protein) can add at both ends of the rod, but do so more slowly. Mechanisms for these processes are discussed. Preliminary results on the binding of the specific hexanucleotide AAGAAG to the disk are given and compared with the known changes on binding nonspecific hexanucleotides or the trinucleotide AAG.

Kinetics↗
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↗
PubMed2006

Dyrk1A phosphorylates alpha-synuclein and enhances intracellular inclusion formation.

Lewy bodies (LBs) are pathological hallmarks of Parkinson disease (PD) but also occur in Alzheimer disease (AD) and dementia of LBs. Alpha-synuclein, the major component of LBs, is observed in the brain of Down syndrome (DS) patients with AD. Dyrk1A, a dual specificity tyrosine-regulated kinase (Dyrk) family member, is the mammalian ortholog of the Drosophila minibrain (Mnb) gene, essential for normal postembryonic neurogenesis. The Dyrk1A gene resides in the human chromosome 21q22.2 region, which is associated with DS anomalies, including mental retardation. In this study, we examined whether Dyrk1A interacts with alpha-synuclein and subsequently affects intracellular alpha-synuclein inclusion formation in immortalized hippocampal neuronal (H19-7) cells. Dyrk1A selectively binds to alpha-synuclein in transformed and primary neuronal cells. Alpha-synuclein overexpression, followed by basic fibroblast growth factor-induced neuronal differentiation, resulted in cell death. We observed that accompanying cell death was increased alpha-synuclein phosphorylation and intracytoplasmic aggregation. In addition, the transfection of kinase-inactive Dyrk1A or Dyrk1A small interfering RNA blocked alpha-synuclein phosphorylation and aggregate formation. In vitro kinase assay of anti-Dyrk1A immunocomplexes demonstrated that Dyrk1A could phosphorylate alpha-synuclein at Ser-87. Furthermore, aggregates formed by phosphorylated alpha-synuclein have a distinct morphology and are more neurotoxic compared with aggregates composed of unmodified wild type alpha-synuclein. These findings suggest alpha-synuclein inclusion formation regulated by Dyrk1A, potentially affecting neuronal cell viability.

Cell Survival↗
PubMed2003

Hyper-expression of small nucleolar RNAs (snoRNAs) in female inflorescences of hazelnut (Corylus avellana L.) supports rRNA aggregation in vitro.

Under certain in vitro (salt and temperature) conditions rRNA aggregation occurs in female inflorescences but not in leaves or pollen RNA preparations from hazelnut (Corylus avellana L.), a species of economic interest. This paper describes experiments addressing an explanation of this phenomenon. The experiments demonstrate that: (i) trans-acting factors induce rRNA aggregate formation in female inflorescences RNA preparations; (ii) these factors support aggregation also of heterologous rRNA; (iii) aggregation is a function of temperature pre-treatment of rRNA and not of source 18S rRNA; (iv) the factors inducing rRNA aggregates are sensitive to RNase; (v) antisense small nucleolar RNAs (snoRNAs) participate in rRNA aggregate formation. snoRNAs are involved in pre-rRNA spacer cleavages, and are required for the two most common types of rRNA modifications: 2'-O-ribose methylation and pseudouridylation. Even though it is questionable whether rRNA aggregation really happens in female inflorescence in vivo, the phenomenon observed in vitro may reflect the abundance of snoRNAs in these reproductive structures. In fact the level of accumulation of three tested snoRNAs, R1, U14 and U3, is much higher in female inflorescence than in leaves or pollen of hazelnut. This finding opens the possibility of studying the role of snoRNAs in tissue development in plants.

Base Sequence↗
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↗