Absence Of RNA Synthesis Research Articles

Determinants of Replication-Fork Pausing at tRNA Genes in Saccharomyces cerevisiae.

Transfer RNA (tRNA) genes are widely studied sites of replication-fork pausing and genome instability in the budding yeast Saccharomyces cerevisiae tRNAs are extremely highly transcribed and serve as constitutive condensin binding sites. tRNA transcription by RNA polymerase III has previously been identified as stimulating replication-fork pausing at tRNA genes, but the nature of the block to replication has not been incontrovertibly demonstrated. Here, we describe a systematic, genome-wide analysis of the contributions of candidates to replication-fork progression at tDNAs in yeast: transcription factor binding, transcription, topoisomerase activity, condensin-mediated clustering, and Rad18-dependent DNA repair. We show that an asymmetric block to replication is maintained even when tRNA transcription is abolished by depletion of one or more subunits of RNA polymerase III. By contrast, analogous depletion of the essential transcription factor TFIIIB removes the obstacle to replication. Therefore, our data suggest that the RNA polymerase III transcription complex itself represents an asymmetric obstacle to replication even in the absence of RNA synthesis. We additionally demonstrate that replication-fork progression past tRNA genes is unaffected by the global depletion of condensin from the nucleus, and can be stimulated by the removal of topoisomerases or Rad18-dependent DNA repair pathways.

Role of Mitochondrial Membrane Spherules in Flock House Virus Replication.

Viruses that generate double-stranded RNA (dsRNA) during replication must overcome host defense systems designed to detect this infection intermediate. All positive-sense RNA viruses studied to date modify host membranes to help facilitate the sequestration of dsRNA from host defenses and concentrate replication factors to enhance RNA production. Flock House virus (FHV) is an attractive model for the study of these processes since it is well characterized and infects Drosophila cells, which are known to have a highly effective RNA silencing system. During infection, FHV modifies the outer membrane of host mitochondria to form numerous membrane invaginations, called spherules, that are ∼50 nm in diameter and known to be the site of viral RNA replication. While previous studies have outlined basic structural features of these invaginations, very little is known about the mechanism underlying their formation. Here we describe the optimization of an experimental system for the analysis of FHV host membrane modifications using crude mitochondrial preparations from infected Drosophila cells. These preparations can be programmed to synthesize both single- and double-stranded FHV RNA. The system was used to demonstrate that dsRNA is protected from nuclease digestion by virus-induced membrane invaginations and that spherules play an important role in stimulating RNA replication. Finally, we show that spherules generated during FHV infection appear to be dynamic as evidenced by their ability to form or disperse based on the presence or absence of RNA synthesis. It is well established that positive-sense RNA viruses induce significant membrane rearrangements in infected cells. However, the molecular mechanisms underlying these rearrangements, particularly membrane invagination and spherule formation, remain essentially unknown. How the formation of spherules enhances viral RNA synthesis is also not understood, although it is assumed to be partly a result of evading host defense pathways. To help interrogate some of these issues, we optimized a cell-free replication system consisting of mitochondria isolated from Flock House virus-infected Drosophila cells for use in biochemical and structural studies. Our data suggest that spherules generated during Flock House virus replication are dynamic, protect double-stranded RNA, and enhance RNA replication in general. Cryo-electron microscopy suggests that the samples are amenable to detailed structural analyses of spherules engaged in RNA synthesis. This system thus provides a foundation for understanding the molecular mechanisms underlying spherule formation, maintenance, and function during positive-sense viral RNA replication.

RNA-related nuclear functions of human Pat1b, the P-body mRNA decay factor.

The evolutionarily conserved Pat1 proteins are P-body components recently shown to play important roles in cytoplasmic gene expression control. Using human cell lines, we demonstrate that human Pat1b is a shuttling protein whose nuclear export is mediated via a consensus NES sequence and Crm1, as evidenced by leptomycin B (LMB) treatment. However, not all P-body components are nucleocytoplasmic proteins; rck/p54, Dcp1a, Edc3, Ge-1, and Xrn1 are insensitive to LMB and remain cytoplasmic in its presence. Nuclear Pat1b localizes to PML-associated foci and SC35-containing splicing speckles in a transcription-dependent manner, whereas in the absence of RNA synthesis, Pat1b redistributes to crescent-shaped nucleolar caps. Furthermore, inhibition of splicing by spliceostatin A leads to the reorganization of SC35 speckles, which is closely mirrored by Pat1b, indicating that it may also be involved in splicing processes. Of interest, Pat1b retention in these three nuclear compartments is mediated via distinct regions of the protein. Examination of the nuclear distribution of 4E-T(ransporter), an additional P-body nucleocytoplasmic protein, revealed that 4E-T colocalizes with Pat1b in PML-associated foci but not in nucleolar caps. Taken together, our findings strongly suggest that Pat1b participates in several RNA-related nuclear processes in addition to its multiple regulatory roles in the cytoplasm.

The stability of abortively cycling T7 RNA polymerase complexes depends upon template conformation.

We have developed a promoter competition assay to determine whether T7 RNA polymerase dissociates from its template during abortive cycling. We find that the stability of the initiation complex (IC) depends upon the conformation of the promoter, and that the degree to which the template is unwound contributes importantly to the stability of the IC. On linear DNA or a relaxed plasmid template, the stability of the IC is very low (t1/2 < 1 min). However, on a supercoiled template, the IC has a stability that is comparable to that of a paused elongation complex (t1/2 = 14 min). At a synthetic promoter that is single stranded in the initiation region (from -5 and downstream), the polymerase forms a highly stable complex (t1/2 > 30 min) even in the absence of RNA synthesis. These findings are important to our understanding of the transition from the IC to an EC.

K252a-potentiation of EGF-induced neurite outgrowth from PC12 cells is not mimicked or blocked by other protein kinase activators or inhibitors

Epidermal growth factor (EGF) has recently been shown to cause certain strains of PC12 cells to extend short neurites. This EGF-induced differentiation of PC12 was found to be potentiated by the protein kinase inhibitor, K252a, in that PC12 cells treated with both EGF and K252a extended long branched neurites similar to those induced by nerve growth factor (NGF). As reported here no other protein kinase inhibitor or activator mimicked or blocked the effect of K252a on EGF-induced PC12 differentiation. Cyclic adenosine 3′,5′-monophosphate (cAMP) also potentiated EGF-induced neurite outgrowth from PC12 cells, but the mechanism of this potentiation was different from that of K252a. Cells that had been exposed to EGF and then stripped of their neurons extended neurites again when retreated with EGF in the absence of RNA synthesis or when treated with NGF in the absence of RNA synthesis. Thus EGF can prime PC12 cells for either EGF or for NGF, a finding that further suggests that EGF and NGF use similar signaling pathways to induced neuronal differentiation of PC12.

Cytoskeleton-bound mRNA for a 40-kDa polypeptide in rat L6 cells is not always translated

The relationship between attachment of mRNA to the cytoskeletal framework and its translation was examined using the mRNA for a polypeptide of 40 kDa (P-40) which is translated in rat L6 myoblasts but not in the myotubes. In both myoblasts and myotubes this mRNA was found to be associated with the cytoskeletal framework. Furthermore, the stability of the association between P-40 mRNA and the cytoskeletal framework in absence of RNA and protein synthesis was examined by using actinomycin D and NaF to block RNA and protein synthesis, respectively. In absence of RNA synthesis portions of both nontranslated P-40 mRNA and translated actin mRNA of myotubes were released into the soluble fraction. In myoblasts, however, both mRNAs remained associated with the cytoskeletal framework following inhibition of RNA synthesis. Inhibition of protein synthesis, on the other hand, had a more dramatic effect on the association between the cytoskeletal framework and P-40 mRNA in myoblasts but not in myotubes. In contrast, the association between actin mRNA and cytoskeletal framework was unaffected by inhibition of protein synthesis in both myoblasts and myotubes. The results of these studies show that the molecular nature of association between cytoskeletal framework and mRNA may differ among mRNAs and may also depend on whether the cells are dividing or are terminally differentiated. Furthermore, no direct relationship between the translation of mRNA and its attachment to the cytoskeletal framework was observed.

The metabolism of ribosomal proteins microinjected into the oocytes of Xenopus laevis

When the total proteins from Xenopus laevis 60 S ribosomal subunits (TP60) were 3H-labeled in vitro and injected back into X. laevis oocytes, most 3H-TP60 are integrated into the cytoplasmic 60 S subunits via the nucleus during 16 h of incubation. In the oocytes whose rRNA synthesis is inhibited, 3H-TP60 are rapidly degraded with a half-life of 2–3 h. This degradation ceased as soon as rRNA synthesis was resumed, suggesting that ribosomal proteins unassociated with nascent rRNA are unstable in the oocytes. The degradation of 3H-TP60 in the absence of RNA synthesis was inhibited by iodoacetamide, a cysteine protease inhibitor, resulting in the accumulation of 3H-TP60 in the nucleus reaching about a threefold concentration in the cytoplasm. Considering the results with enucleated oocytes, we suggest that the X. laevis nucleus has a limited capacity to accumulate ribosomal proteins in an active manner but that those ribosomal proteins accumulated in excess over rRNA synthesis are degraded by a cysteine protease in the nucleus. By contrast, ribosomal proteins from Escherichia coli only equilibrate between the nucleus and the cytoplasm and are degraded by serine protease(s) in the cytoplasm without being integrated in the form of ribosomes in the nucleus.

Leishmanial differentiation [formula omitted]: Induction of heat shock proteins

During temperature-induced in vitro differentiation from the promastigote to the amastigote form of the parasitic protozoan Leishmania mexicana , seven actively synthesized proteins were identified. These proteins corresponded precisely in molecular weight to the well-known heat shock proteins seen in species such as Drosophila . The minimal DNA synthesis observed in the differentiating parasites indicated that little cell division occurs during this process. The absence of RNA synthesis during early temperature-induced differentiation suggests that the leishmanial heat shock proteins are post-transcriptionally regulated. Heat shock proteins may play an adaptive role in the transition of Leishmania from arthropod to mammalian host.

The synthesis and turnover of ferritin in rat L-6 cells. Rates and response to iron, actinomycin D, and desferrioxamine.

Rates of ferritin accumulation in the L-6 line of rat skeletal myoblasts cultured in the presence of ferric nitrilotriacetate (FeNTA) were measured and found to vary with the extracellular concentration of FeNTA as predicted from dose response experiments. The rate of ferritin accumulation is constant for up to 92 h in these cells after addition of iron, with the exception of the first few hours of synthesis in which the rate is approximately twice that observed at later times. Experiments in which the specific activity of newly synthesized ferritin was calculated implied that the rate of ferritin degradation might be higher in this earlier period as well; pulse-chase experiments confirmed this hypothesis. Ferritin synthesis is thought to be induced by iron in the absence of RNA synthesis. Accordingly, actinomycin D was shown not to inhibit the synthesis of ferritin in response to FeNTA. Rather, a pronounced stimulation of the synthetic rate was observed. Finally, desferrioxamine was shown both to decrease the rate of ferritin synthesis and increase its rate of degradation. Possible mechanisms for these phenomena are discussed.

Hormonal regulation of glucokinase in primary cultures of rat hepatocytes.

Glucokinase activity in rat hepatocyte cultures declined with a half-time, t1/2, of 32 h during 3 days under serum-free conditions. Addition of insulin and triamcinolone to the culture medium prevented this decay. Glucokinase levels in hepatocytes derived from fasted rats could be elevated from 7.4 to 16.4 mU/mg protein in the presence of insulin and triamcinolone. In 2-day-old cultures glucokinase was induced in the presence of both hormones with a half time, t1/2, of 5.1 h. In cultures treated for 2 days with triamcinolone, insulin induced a 80% increase of glucokinase even in the absence of glucocorticoids. Insulin induction was dependent on protein synthesis but occurred in the absence of RNA synthesis. Glucocorticoid action, however, depended on RNA synthesis suggesting that glucocorticoids control transcription. Insulin evoked half-maximal effects at 3 nM and dexamethasone and triamcinolone at 0.1 and 1 nM respectively. Degradation of glucokinase was initiated in 2-day-old hepatocytes after removal of triamcinolone and insulin. Protein synthesis was essential for the onset of degradation and glucagon did not affect the rate of glucokinase degradation.

Alterations in tRNAs Containing 2‐Methylthio‐N6‐(Δ2‐isopentenyl)‐adenosine during Growth of Enteropathogenic Escherichia coli in the Presence of Iron‐Binding Proteins

Escherichia coli grown in chemically produced iron-deficient media have well characterized alterations in the chromatographic properties of tRNAs containing the modified nucleoside 2-methylthio-N6-(delta2-isopentenyl) adenosine. The present report shows that similar tRNA alterations occur in enteropathogenic E. coli inhibited by human milk and bovine colostrum, the inhibited bacteria containing 10% or less of the normal tRNA species. Adding sufficient iron to saturate the iron-binding capacity of the lactoferrin present in milk and colostrum reversed these changes which are probably due to a failure to methylthiolate the isopentenyladenosine. Although adding iron led to a rapid replacement of abnormal tRNA by the chromatographically normal species, and to a resumption of multiplication, the tRNA alterations are not directly related to the inhibition of growth. Strains of E. coli which grew normally in milk, colostrum and in defined media containing the iron-binding protein transferrin or ovotransferrin also contained about 90% of the abnormal species. Rapid conversion of abnormal tRNA to normal tRNA occurred on adding iron and in the absence of RNA synthesis. The tRNA changes are discussed in relation to their possible connection with both the adaptation of E. coli to growth under the iron-restricted conditions imposed by iron-binding proteins in tissue fluids and with bacterial pathogenicity.

The synthesis of RNA and protein by nongerminating zygotes of the aquatic fungus, Allomyces macrogynus

Zygotes of Allomyces macrogynus, maintained under conditions that do not support their germination, synthesize both protein and RNA, although at a lower rate than germinating cells. At least eight different proteins are synthesized by nongerminating zygotes. The predominant class of RNA synthesized is 4 S in size. In addition, it appears that some messenger RNAs are also transcribed because there is a selective inhibition of the synthesis of some proteins in the absence of RNA synthesis.

Deinduction of phosphoenolpyruvate carboxykinase (guanosine triphosphate) synthesis in Reuber H-35 cells.

The activity of phosphoenolpyruvate carboxykinase (GTP) in Reuber H-35 cells was decreased after the removal of 6-N,2-O-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) from the medium. The decrease in activity was shown immunochemically to be the result of a rapid cessation in specific enzyme synthesis, occurring with a half-time of 40 min. The removal of dexamethasone, a less potent inducer of the enzyme in these cells, did not effect the activity of P-enolpyruvate carboxykinase or its rate of synthesis. Insulin added to either dibutyryl cyclic AMP or dexamethasone-treated cells produced a decline in specific enzyme synthesis which was not as rapid as that observed upon removal of dibutyryl cyclic AMP. This effect of insulin did not require the presence of glucose in the culture medium. Estimates of the half-life of the mRNA for P-enolpyruvate carboxykinase using actinomycin D and cordycepin suggested that after the inhibition of transcription of mRNA, enzyme synthesis continued for periods considerably longer than that observed after deinduction caused by removal of dibutyryl cyclic AMP. In addition, the synthesis of the enzyme could be restimulated by dibutyryl cyclic AMP in the absence of RNA synthesis. It was proposed that the deinduction of phosphoenolpyruvate carboxykinase in these cells is being regulated at the post-transcriptional or translational level.

Cytochemical studies on the protamine-type protein transition in sperm nuclei after fertilization and the early embryonic histones of Urechis caupo

Cytochemical staining characteristics of nuclear histones during postfertilization maturation division and various early embryonic stages in Urechis have been studied. The transition of protamine-type protein to adult histones in the sperm nucleus is accomplished by 15 min after entrance into the egg cytoplasm. Newly synthesized egg proteins migrate into enlarging male and female pronuclei after this transition, followed by pronuclear DNA synthesis and fusion. The shift from protamine-type protein to adult histones, which occurs in the absence of RNA synthesis during the postfertilization maturation division of the egg, may be one of the processes involved in the normal structural reorganization of chromosomes. Such a reorganization is likely to be a prerequisite for chromosome replication and mitosis. No qualitative differences are detected in the stainability of histones of unfertilized eggs and embryos at the cleavage and later stages of development.

Effect of actinomycin D, pancreozymin and secretin on RNA synthesis and protein synthesis measured in vivo in rat pancreas

As has been reported earlier (Reggio, H., Cailla-Deckmyn, H. and Marchis-Mouren, G. (1971) J. Cell Biol. 50, 333–343) pancreozymin enhances not only enzymatic secretion but also protein synthesis in rat pancreas in vivo. We inhibit RNA synthesis by actinomycin D and we measure the stimulation of protein synthesis both in the presence and absence of RNA synthesis: stimulation yields are similar. Consequently it appears that the transcription step is not involved in the stimulation of protein synthesis by pancreozymin.

Protein synthesis during germination of Peronospora tabacina (Adam) conidia

The characteristics of an in vitro amino acid-incorporating system prepared from germinated Peronospora tabacina conidia are described. Material sedimenting at 20,000 g and at 117,000 g was found to possess significant amino acid-incorporating activity. Although germination is accompanied by an increase in the activity of both these fractions, if RNA synthesis is inhibited during germination, activity increases only in the 20,000 g fraction. Since germ tube formation occurs in the absence of RNA synthesis, it is suggested that the protein required for germ tube formation is largely synthesized by this 20,000 g fraction. The increase in amino acid-incorporating activity in this fraction begins during the preparation of conidia at 0o) and prior to the start of incubation at 15 o and may result from the removal of some factor during washing.

Cell fusion by Newcastle disease virus in the absence of RNA synthesis.

OUR studies on the cytopathic effects of Newcastle disease virus (NDV) grown in chick embryo fibroblast cell cultures have shown that the principal cytopathic effect involves the formation of polykaryocytes by cell fusion (our unpublished work). This ability is related directly to the virulence of the infecting strain; those pathogenic for chick embryos readily induce cell fusion whereas avirulent strains induce little or no fusion1. We now report that, although protein synthesis is required for NDV-induced cell fusion, RNA synthesis is not. Furthermore, blocking of RNA synthesis significantly increases cell fusion by avirulent strains.

Translation-level control of amylase and protein synthesis by epinephrine.

The synthesis of RNA, protein, and amylase has been studied in normal and epinephrine-treated slices of rat parotid gland. Epinephrine stimulates synthesis of total cell protein and specifically of amylase, but amylase synthesis is preferentially increased. There is no change in either the specific radioactivity or pattern of labeling of total RNA. In the presence of actinomycin D, epinephrine stimulates secretion of amylase, as well as protein and amylase synthesis. RNA synthesis under these conditions is reduced to 10 per cent of control values. Slices obtained from animals treated with actinomycin D nine and a half hours prior to study show responses to epinephrine comparable to those from control animals, with RNA synthesis inhibited by 70 per cent. Since amylase synthesis is stimulated by epinephrine in the absence of RNA synthesis, control of the production of this enzyme is probably exerted at the level of translation.

The control of haemoglobin synthesis: factors controlling the output of alpha and beta chains.

Analysis of the effects of amino acid starvation in reticulocytes is comparatively simple compared with similar analysis in other tissues of whole organisms. This is mainly because of the absence of RNA synthesis in reticulocytes, but also because the bulk of the protein being synthesized is haemoglobin, a protein whose structure is completely known. The absence of RNA synthesis eliminates complications that would otherwise arise through RNA-mediated control mechanisms which in turn might mask the effects of amino acid starvation on the protein synthetic machinery in the cells (Munro, 1969). Consequently reticulocytes have been used to study the effect of amino acid starvation on the actual process of protein synthesis and assembly.

Replication of RNA bacteriophage R23. I. Quantitative aspects of phage RNA and protein synthesis.

Abstract Infection of Escherichia coli by R23, a newly isolated strain of RNA bacteriophage, has been found markedly to suppress synthesis of bacterial RNA. Approximately 60% of total RNA synthesized after infection was encapsulated in progeny phage particles. In contrast, after infection with the RNA phages f2 or Qβ, only 25 to 30% of newly synthesized RNA was phage RNA. Thirty to 40% of the RNA synthesized after B23 infection was characterized as a second form of phage-specific RNA by its resistance to RNase. Synthesis of bacterial proteins was also markedly inhibited after infection by R23 and ultimately replaced by phage-directed protein synthesis. B23 “early” protein synthesis, detected as phage RNA polymerase activity, was evident five to ten minutes after infection and increased until 20 to 30 minutes. In contrast, in the normally infected cell, synthesis of coat protein was predominant at a later time, beginning 30 to 45 minutes after infection and reaching a maximum between 45 and 75 minutes of infection, when coat protein constituted almost all of the newly synthesized protein. Coat protein synthesis was not detectable in the absence of RNA synthesis, whereas phage RNA polymerase induction required little or no RNA synthesis.