Requires RNA Synthesis Research Articles

RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro.

Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus Orthocurvulavirus within the family Orthocurvulaviridae. The purpose of the study was to determine the biochemical requirements for RNA synthesis catalyzed by HetRV6 RNA-dependent RNA polymerase (RdRp). HetRV6 RdRp was expressed in Escherichia coli and isolated to near homogeneity using liquid chromatography. The enzyme activities were studied in vitro using radiolabeled UTP. The HetRV6 RdRp was able to initiate RNA synthesis in a primer-independent manner using both virus-related and heterologous single-stranded (ss)RNA templates, with a polymerization rate of about 46 nt/min under optimal NTP concentration and temperature. NTPs with 2′-fluoro modifications were also accepted as substrates in the HetRV6 RdRp-catalyzed RNA polymerization reaction. HetRV6 RdRp transcribed viral RNA genome via semi-conservative mechanism. Furthermore, the enzyme demonstrated terminal nucleotidyl transferase (TNTase) activity. Presence of Mn2+ was required for the HetRV6 RdRp catalyzed enzymatic activities. In summary, our study shows that HetRV6 RdRp is an active replicase in vitro that can be potentially used in biotechnological applications, molecular biology, and biomedicine.

Live and let die: ZBP1 senses viral and cellular RNAs to trigger necroptosis.

Necroptosis is a programmed form of inflammatory cell death involved in various pathologies, such as viral infections. In two new papers published in The EMBO Journal and EMBO Reports , Z‐DNA binding protein 1 (ZBP1) is now shown to sense RNAs during viral infection or after caspase inhibition and activate necroptosis. This may suggest that Z‐RNAs are molecular patterns for activation of necroptosis.

Sensing of viral and endogenous RNA by ZBP1/DAI induces necroptosis

Nucleic acids are potent triggers for innate immunity. Double‐stranded DNA and RNA adopt different helical conformations, including the unusual Z‐conformation. Z‐DNA/RNA is recognised by Z‐binding domains (ZBDs), which are present in proteins implicated in antiviral immunity. These include ZBP1 (also known as DAI or DLM‐1), which induces necroptosis, an inflammatory form of cell death. Using reconstitution and knock‐in models, we report that mutation of key amino acids involved in Z‐DNA/RNA binding in ZBP1's ZBDs prevented necroptosis upon infection with mouse cytomegalovirus. Induction of cell death was cell autonomous and required RNA synthesis but not viral DNA replication. Accordingly, ZBP1 directly bound to RNA via its ZBDs. Intact ZBP1‐ZBDs were also required for necroptosis triggered by ectopic expression of ZBP1 and caspase blockade, and ZBP1 cross‐linked to endogenous RNA. These observations show that Z‐RNA may constitute a molecular pattern that induces inflammatory cell death upon sensing by ZBP1.

Transcription Start Site Scanning and the Requirement for ATP during Transcription Initiation by RNA Polymerase II

Saccharomyces cerevisiae RNA polymerase (Pol) II locates transcription start sites (TSS) at TATA-containing promoters by scanning sequences downstream from the site of preinitiation complex formation, a process that involves the translocation of downstream promoter DNA toward Pol II. To investigate a potential role of yeast Pol II transcription in TSS scanning, HIS4 promoter derivatives were generated that limited transcripts in the 30-bp scanned region to two nucleotides in length. Although we found that TSS scanning does not require RNA synthesis, our results revealed that transcription in the purified yeast basal system is largely ATP-independent despite a requirement for the TFIIH DNA translocase subunit Ssl2. This result is rationalized by our finding that, although they are poorer substrates, UTP and GTP can also be utilized by Ssl2. ATPγS is a strong inhibitor of rNTP-fueled translocation, and high concentrations of ATPγS make transcription completely dependent on added dATP. Limiting Pol II function with low ATP concentrations shifted the TSS position downstream. Combined with prior work, our results show that Pol II transcription plays an important role in TSS selection but is not required for the scanning reaction.

In Pursuit of Genes of Glucose Metabolism

If you don't know where you're going, you might wind up someplace else—Yogi Berra My professional journey, once I accepted the fact that I would not play first base for the Chicago Cubs, was not accomplished in a straight line. I entered medical school intending to become a family physician but soon became interested in research. I took an internal medicine residency at the University of California, San Francisco (UCSF), then a long research fellowship in the nascent field of molecular biology at the National Institutes of Health (NIH), and followed that with training in clinical endocrinology at the University of Wisconsin. I spent the next fifteen years dividing my time between the clinic and laboratory at the University of Iowa. Finally, I decided to concentrate on research and spent the next twenty-three years at Vanderbilt University. For the past eleven years, I have been at the University of Iowa, where I helped establish a new diabetes research center. My research journey also had several different directions. Upon reflection, I guess I ended up “someplace else” by choice, and it certainly has been an interesting trip. It is one I unreservedly recommend.

Na+/K+‐ATPase inhibitor palytoxin enhances vulnerability of cultured cerebellar neurons to domoic acid via sodium‐dependent mechanisms

Dysfunction or deficiency of the Na(+)/K(+)-ATPase appears to be a common event in a variety of pathological conditions in the central nervous system. Studies on neurotoxicity associated to impaired Na(+)/K(+)-ATPase activity have focused on NMDA receptors, while the involvement of non-NMDA receptors has been much less explored. We show that mild, non-toxic, exposures to the Na(+)/K(+)-ATPase inhibitor palytoxin (PTX) synergistically sensitized the vulnerability of neurons to normally non-toxic concentrations of domoic acid, leaving NMDA receptor-mediated excitotoxic response unaltered. Enhancement of excitotoxicity required at least 1 h pre-exposure to PTX, was not observed after longer exposures to PTX, and did not require RNA synthesis. PTX caused a voltage-sensitive Na(+) channel-independent increase in intracellular Na(+). Both intracellular Na(+) increase and potentiation of excitotoxicity depended upon the external concentrations of Na(+) and Cl(-), and were suppressed by the anion exchanger blocker 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid in a dose-dependent manner. Other stilbene derivatives, chloride channel antagonists or Na(+) cotransporter inhibitors proved ineffective. Our results demonstrate a crucial role for Na(+)/K(+)-ATPase activity in determining neuronal vulnerability to domoic acid-mediated excitotoxicity. They also raise reasonable concern about possible risks for human health associated to the ingestion of low amounts of phycotoxins PTX and domoic acid in food.

The roles of RNA synthesis and protein translation during reconsolidation of passive-avoidance learning in the day-old chick

This series of experiments investigated the role of protein translation and RNA synthesis on consolidation and reconsolidation of passive avoidance learning (PAL) in day-old chicks. Although it is well established that protein translation is required after a reminder, there are conflicting reports in the literature concerning the requirement for RNA synthesis at this time. Day-old male New Hampshire × White Leghorn chicks were trained on a single trial passive avoidance task. The results confirmed the requirement for protein translation during reconsolidation with memory deficits induced by anisomycin (ANI) (10 mg/kg) detected at 60 min post-reminder. It was also established that RNA synthesis was required for consolidation of PAL through inhibition by 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) (0.075 µg/kg), administered at or after training. The same dose of DRB was also found to inhibit memory post-reactivation. However injections were required before the reminder trial and memory deficits were evident by 60 min, consistent with that found for ANI post-reminder. As with ANI, the DRB-induced memory deficit post-reminder was also transient, and resolved by 24 h post-reminder. For both reconsolidation drug studies, the memory deficit was wholly dependent on the memory being reactivated by a reminder-trial. The study highlights an important role for RNA synthesis following memory reactivation.

Botrytis cinerea isolates collected from grapes present different requirements for conidia germination

Botrytis cinerea presents high variability in several biological traits, which can be explained by the high degree of genotypic diversity among isolates. Because this genetic variability might be related to phenotypic differences the requirements for conidia germination of three natural isolates (G1, G5 and G11) obtained from grapes and belonging to the same genetic group were analyzed. The results showed that contact with a solid surface was a common requisite for conidia germination of the isolates but they differed in their nutritional requirements to germinate. Isolate G11 was able to germinate in the absence of a carbon or nitrogen source. G1 and G5 required the presence of a carbon source such as glucose, fructose or sucrose. In G11 and G5 isolates a much higher rate of germination was obtained in the presence of sucrose.It was shown with a pharmacological approach that the cAMP stimulated the germination only in those isolates requiring a carbon source. Conidia germination of G1 and G5 was inhibited by EGTA, a calcium chelator. Isolate G11 germinated in the presence of this compound. On the other hand the germination of three B. cinerea isolates required protein synthesis and did not require RNA synthesis. To explain the ability of isolate G11 to germinate in water the content of total and reducing sugars, mannitol/l-arabitol, trehalose, and proteins in the nongerminated conidia of the three isolates was compared. The isolates presented similar amounts of total and reducing sugars. In the three isolates the amount of mannitol/l-arabitol was higher than that of trehalose. In isolate G11 total protein content was twice higher than in the other isolates.

Abortive Initiation and Productive Initiation by RNA Polymerase Involve DNA Scrunching

Using single-molecule DNA nanomanipulation, we show that abortive initiation involves DNA "scrunching"--in which RNA polymerase (RNAP) remains stationary and unwinds and pulls downstream DNA into itself--and that scrunching requires RNA synthesis and depends on RNA length. We show further that promoter escape involves scrunching, and that scrunching occurs in most or all instances of promoter escape. Our results support the existence of an obligatory stressed intermediate, with approximately one turn of additional DNA unwinding, in escape and are consistent with the proposal that stress in this intermediate provides the driving force to break RNAP-promoter and RNAP-initiation-factor interactions in escape.

Native Replication Intermediates of the Yeast 20 S RNA Virus Have a Single-stranded RNA Backbone

20 S RNA virus is a positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome (2.5 kb) only encodes its RNA polymerase (p91) and forms a ribonucleoprotein complex with p91 in vivo. A lysate prepared from 20 S RNA-induced cells showed an RNA polymerase activity that synthesized the positive strands of viral genome. When in vitro products, after phenol extraction, were analyzed in a time course, radioactive nucleotides were first incorporated into double-stranded RNA (dsRNA) intermediates and then chased out to the final single-stranded RNA products. The positive and negative strands in these dsRNA intermediates were non-covalently associated, and the release of the positive strand products from the intermediates required a net RNA synthesis. We found, however, that these dsRNA intermediates were an artifact caused by phenol extraction. Native replication intermediates had a single-stranded RNA backbone as judged by RNase sensitivity experiments, and they migrated distinctly from a dsRNA form in non-denaturing gels. Upon completion of RNA synthesis, positive strand RNA products as well as negative strand templates were released from replication intermediates. These results indicate that the native replication intermediates consist of a positive strand of less than unit length and a negative strand template loosely associated, probably through the RNA polymerase p91. Therefore, W, a dsRNA form of 20 S RNA that accumulates in yeast cells grown at 37 degrees C, is not an intermediate in the 20 S RNA replication cycle, but a by-product.

Transcriptional program of apoptosis induction following interleukin 2 deprivation: identification of RC3, a calcium/calmodulin binding protein, as a novel proapoptotic factor.

Apoptosis of mature T lymphocytes preserves immune system homeostasis by counteracting transient increases in T-cell number. This process is regulated, at least in part, by the cytokine interleukin 2 (IL-2): T cells deprived of IL-2 undergo apoptosis. The mechanism of apoptosis induction by IL-2 deprivation remains to be determined but is known to require RNA synthesis, implying the existence of transcriptionally activated genes whose products induce cell death. To identify such genes, we have performed expression profiling in IL-2-dependent T cells following cytokine deprivation. Our results reveal an intricate transcriptional program entailing the induction of known proapoptotic factors and the simultaneous repression of known antiapoptotic factors. Surprisingly, one gene whose transcription substantially increased was RC3 (also called neurogranin), which encodes a calmodulin binding protein thought to be a neural-specific factor involved in learning and memory. We show that ectopic expression of RC3 in IL-2-dependent T cells increases the intracellular Ca(2+) concentration and induces apoptosis even in the presence of cytokine. Buffering the Ca(2+) increase with the cytoplasmic Ca(2+) chelator BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N1,N-tetraacetic acid] blocks RC3-induced apoptosis, indicating that the rise in intracellular Ca(2+) is required for apoptotic death. RC3 mutants unable to bind calmodulin fail to increase intracellular Ca(2+) levels and to induce apoptosis. Based upon these results, we propose that IL-2 deprivation raises the level of RC3 and other apoptotic factors, which induce apoptosis by increasing the intracellular Ca(2+) concentration.

Antihistamine terfenadine potentiates NMDA receptor-mediated calcium influx, oxygen radical formation, and neuronal death

We previously reported that the histamine H1 receptor antagonist terfenadine enhances the excitotoxic response to N-methyl- d-aspartate (NMDA) receptor agonists in cerebellar neurons. Here we investigated whether this unexpected action of terfenadine relates to its antihistamine activity, and which specific events in the signal cascade coupled to NMDA receptors are affected by terfenadine. Low concentrations of NMDA (100 μM) or glutamate (15 μM) that were only slightly (<20%) toxic when added alone, caused extensive cell death in cultures pre-exposed to terfenadine (5 μM) for 5 h. Terfenadine potentiation of NMDA receptor response was mimicked by other H1 antagonists, including chlorpheniramine (25 μM), oxatomide (20 μM), and triprolidine (50 μM), was prevented by histamine (1 mM), and did not require RNA synthesis. Terfenadine increased NMDA-mediated intracellular calcium and cGMP synthesis by approximately 2.4 and 4 fold respectively. NMDA receptor-induced cell death in terfenadine-treated neurons was associated with a massive production of hydrogen peroxides, and was significantly inhibited by the application of either (+)-alpha-tocopherol (200 μM) or the endogenous antioxidant melatonin (200 μM) 15 min before or up to 30 min after receptor stimulation. This operational time window suggests that an enduring production of reactive oxygen species is critical for terfenadine-induced NMDA receptor-mediated neurodegeneration, and strengthens the importance of antioxidants for the treatment of excitotoxic injury. Our results also provide direct evidence for antihistamine drugs enhancing the transduction signaling activated by NMDA receptors in cerebellar neurons.

Elevated perfusion pressure upregulates endothelin-1 and endothelin B receptor expression in the rabbit carotid artery.

To investigate the hypothesis that high blood pressure activates the endothelin system in the vessel wall, isolated segments of the rabbit carotid artery were subjected to different levels of perfusion pressure. Both preproendothelin-1 (ppET-1) mRNA abundance and intravascular ET-1 peptide content were strongly upregulated on raising the intraluminal pressure from 90 to 160 mm Hg for 3 to 12 hours, and this increase in ppET-1 mRNA occurred predominantly in the endothelial cells. Endothelin-converting enzyme-1 and endothelin A receptor (ET(A)-R) expression were pressure-insensitive, whereas that of the ET(B)-R in the smooth muscle cells was also significantly enhanced. Both the pressure-induced increase in ppET-1 and ET(B)-R expression required RNA synthesis because they were abolished by actinomycin D. The nuclear signaling mechanisms involved therein, however, appeared to be different. Thus, the pressure-induced expression of ppET-1 and activation of CCAAT-enhancer binding proteins beta and delta were blocked by the tyrosine kinase inhibitor herbimycin A, whereas ET(B)-R expression and the nuclear translocation of activator protein-1 were abolished by the protein kinase C inhibitor Ro 31-8220. One consequence of these presumably deformation-induced changes in gene expression was an increased rate of apoptosis of the smooth muscle cells in the media that if transferable to the situation in human blood vessels may contribute to hypertension-induced arterial remodeling.

The light induction of maize phosphoenolpyruvate carboxylase kinase translatable mRNA requires transcription but not translation

We have previously demonstrated that the level of translatable mRNA for phosphoenolpyruvate carboxylase kinase in maize leaves is increased in response to light (Hartwell et al. 1996; Plant Journal10, 1071–1078). To identify the steps required for this increase, we have examined the effects of protein and RNA synthesis inhibitors. The RNA synthesis inhibitors actinomycin D and cordycepin (500 μM) strongly inhibited the light‐induced increases in kinase translatable mRNA and the apparent phosphorylation state of phosphoenolpyruvate carboxylase, as judged by its sensitivity to inhibition by L‐malate. The protein synthesis inhibitors cycloheximide and puromycin blocked the light‐induced increase in the apparent phosphorylation state of phosphoenolpyruvate carboxylase but not the increase in kinase translatable mRNA. Indeed, the amount of phosphoenolpyruvate carboxylase kinase translatable mRNA after 3 h of illumination of leaves treated with either 1 mM puromycin or 100 μM cycloheximide was double that in illuminated control leaves. Each inhibitor reduced the light‐induction of two control genes, malic enzyme and pyruvate, phosphate dikinase. Thus the light induction of phosphoenolpyruvate carboxylase kinase translatable mRNA requires RNA synthesis, but not protein synthesis.

Identification of interleukin-6 as a factor that induces neurite outgrowth by PC12 cells primed with NGF.

We have screened for a factor that induces neurite outgrowth by PC12 cells only after NGF pretreatment in the supernatants of 38 transformed cell lines, finding three positive clones. The factor showing the strongest activity was purified and identified as IL6. In this NGF-IL6 system, the process of neurite outgrowth by PC12 cells can be separated into NGF-dependent and IL6-dependent steps. The IL6-dependent step requires RNA synthesis, suggesting that IL6 induces new gene expression depending on NGF-priming. These findings suggest that the gene expression during the differentiation process is regulated by at least two signals.

Apoptosis in cerebellar granule cells is blocked by high KCl, forskolin, and IGF-1 through distinct mechanisms of action: the involvement of intracellular calcium and RNA synthesis

Cerebellar granule cells deprived of depolarizing concentration of extracellular potassium, [K+]o, undergo apoptosis. We here report that this apoptotic process is associated with an immediate and permanent decrease in the levels of free intracellular calcium, [Ca2+]i. Although forskolin and IGF-1 are both able to prevent apoptosis, only forskolin is able to counteract the instantaneous decrease of [Ca2+]i. However, the early effect of forskolin on [Ca2+]i is lost after longer incubation in low [K+]o. The calcium antagonist nifedipine is able to inhibit the survival effect of high [K+]o, while not affecting forskolin and IGF-1 promoted survival, as assessed by viability and genomic DNA analysis. Accordingly, the L-type calcium channels agonist Bay K8644 significantly enhanced the survival of low KCl treated neurons. To temporally characterize the signal transduction events and the essential transcriptional step in cerebellar granule cells apoptosis, we determined the time course of the rescue capacity of high [K+]o, forskolin, IGF-1, and actinomycin D. Addition of high KCl, forskolin, or IGF-1 6 hr after the initial KCl deprivation saves 50% of cells. Remarkably, 50% of neurons loss the potential to be rescued by actinomycin D after only 1 hr in low [K+]o. Finally, we show that the survival promoting activities of high [K+]o, forskolin, and IGF-1 do not require RNA synthesis. We conclude that [Ca2+]i is involved in the survival promoting activity exerted by high [K+]o but not in those of forskolin and IGF-1, and that all three agents, although rescuing neurons from apoptosis through distinct mechanisms of action, do not necessitate RNA transcription.

MODULATION OF PROLIFERATION AND CELLULAR-CHANGES OF HUMAN PROSTATIC-CANCER CELLS BY ACTIVATED LYMPHOCYTES

The effect of immune cells on the growth and development of human prostatic cancer cells was investigated. Cell proliferation of androgen-independent human prostatic cancer cells JCA-1 was reduced 30-40% with the supplementation of a growth modulating activity present in lymphocyte conditioned medium. The slower growing cells had a longer doubling time revealing a blocking for G(1) cells entering into S phase. The clonogenicity of the cells was also reduced. Displayed in parallel was altered phenotypes including the development of dendrite-like processes with less intercellular contact. Immunocytochemical staining showed significantly increased: expression of actin, vimentin and cytokeratin 8, suggesting a differentiated cell type. Activated lymphocytes were shown to require RNA synthesis for providing the modulating activity which was estimated to be 18,500-27,500 Da. The role of immune cells in growth regulation of prostatic cells is discussed.

K252a potentiates epidermal growth factor-induced differentiation of PC12 cells.

Epidermal growth factor (EGF) induced short neurites in two different strains of PC12 cells. The length of the EGF-induced neurites was markedly increased in the presence of the protein kinase inhibitor K252a, which is known to inhibit differentiation induced by nerve growth factor (NGF). EGF-induced differentiation of PC12 required RNA synthesis and activity of the ras proto-oncogene product. EGF increased the levels of three neurofilament proteins and the mRNA level of two late response genes (SCG10 and 63) known to be induced by NGF. Together, EGF and K252a caused a greater increase in these mRNAs than did either agent alone. K252a did not alter the extent of EGF-induced autophosphorylation of the EGF receptor, but it did decrease the extent of receptor phosphorylation in the absence of added EGF. Thus, the ability of the EGF receptor to trigger neuronal differentiation may depend on the state of its phosphorylation at serine and/or threonine residues. Two other strains of PC12 did not extend neurites when exposed to EGF, even when K252a was also present. Thus, the differentiating effect of EGF on PC12 is PC12 strain-specific.

Regulation of DNA replication in vitro by the transcriptional activation domain of GAL4-VP16.

Studies of DNA viruses have provided evidence that eukaryotic transcriptional activator proteins can enhance the efficiency of DNA replication as well as transcription. The mechanism of this effect was studied in vitro using the chimeric transcription factor GAL4-VP16 and a DNA template containing GAL4 binding sites adjacent to the simian virus 40 origin of DNA replication. The binding of GAL4-VP16 prevented the repression of DNA replication which otherwise occurred when the template was assembled into chromatin. Relief of repression by GAL4-VP16 required both its DNA-binding and transcriptional activation domains but did not require RNA synthesis. The results are consistent with a general model in which transcriptional activators stimulate eukaryotic DNA replication by modifying the outcome of the competition between initiation factors and histones for occupancy of the origin.

Involvement of Fis protein in replication of the Escherichia coli chromosome

We report evidence indicating that Fis protein plays a role in initiation of replication at oriC in vivo. At high temperatures, fis null mutants form filamentous cells, show aberrant nucleoid segregation, and are unable to form single colonies. DNA synthesis is inhibited in these fis mutant strains following upshift to 44 degrees C. The pattern of DNA synthesis inhibition upon temperature upshift and the requirement for RNA synthesis, but not protein synthesis, for resumed DNA synthesis upon downshift to 32 degrees C indicate that synthesis is affected in the initiation phase. fis mutations act synergistically with gyrB alleles known to affect initiation. oriC-dependent plasmids are poorly established and maintained in fis mutant strains. Finally, purified Fis protein interacts in vitro with sites in oriC. These interactions could be involved in mediating the effect of Fis on DNA synthesis in vivo.