Post-transcriptional Steps Research Articles

The anti-inflammatory drug sodium salicylate inhibits nitric oxide formation induced by interleukin-1β at a translational step, but not at a transcriptional step, in hepatocytes

Recent evidence suggests that nitric oxide (NO) mediates cellular injury under the pathological conditions such as endotoxemia in the liver of rats. Regulation of NO production is crucial for improving the hepatic dysfunction. We have previously reported that, in cultured rat hepatocytes, a single cytokine interleukin-1β (IL-1β) stimulated a release of nitrite, an oxidation product of NO, into culture medium dose- and time-dependently. The objective of this study was to investigate an ability of the anti-inflammatory drug NaSA to affect the production of NO in hepatocytes. IL-1β increased levels of inducible NO synthase (iNOS) messenger RNA (mRNA) with a maximal effect at 8 hours in primary cultures of rat hepatocytes. Nuclear factor-κB (NF-κB), that is an important nuclear factor protein in iNOS gene transcription in response to inflammatory mediators, also appeared in the nuclear fraction of hepatocytes 1 hour after addition of IL-1β. Sodium salicylate markedly inhibited the NO formation induced by IL-1β, but did not affect NF-κB activation and iNOS mRNA induction. Western blot analysis revealed that sodium salicylate (NaSA) blocked a step of iNOS protein synthesis. These findings indicate that NaSA may reduce hepatic injury by preventing the induction of NO formation in response to IL-1β at the posttranscriptional step.(Hepatology 1997 Feb;25(2):416-20)

Multiple Transcripts for Rat Nucleoside Diphosphate Kinase α Isoform Are Structurally Categorized into Two Groups That Exhibit Cell-specific Expression and Distinct Translation Potential

Rat nucleoside diphosphate (NDP) kinase is composed of two isoforms (alpha and beta) encoded by independent genes. The mRNAs are expressed ubiquitously; however, the level of expression is tissue-dependent and is also up- or down-regulated under certain conditions, including growth stimulation, differentiation, and tumor metastasis. To address the regulatory mechanisms of gene expression for the rat NDP kinase major isoform alpha (an nm23-H2/PuF homologue), we identified the transcription initiation sites in detail by RNase protection and 5'-rapid amplification of DNA ends and located the core promoter region by chloramphenicol acetyltransferase assay. The transcripts, initiated from an extraordinarily wide range of sites, were categorized into two groups; one transcribed from an upstream region was spliced in the untranslated region (group 1), whereas the other initiated in the downstream region was not (group 2). RNase protection demonstrated that the group 1 mRNA was the dominant form present in all tissues except heart and skeletal muscle. In situ hybridization revealed cell-specific expression of these mRNA species. Furthermore, they differed in the translational efficiency (the group 2 alpha > beta > the group 1 alpha). These findings suggest that the regulation of the NDP kinase expression at both transcriptional and posttranscriptional steps could be fundamentally governed by the selection of transcription initiation sites.

The Anti–Inflammatory Drug Sodium Salicylate Inhibits Nitric Oxide Formation Induced by Interleukin–1β At A Translational Step, But Not At A Transcriptional Step, in Hepatocytes

ABSTRACT Recent evidence suggests that nitric oxide (NO) mediates cellular injury under the pathological conditions such as endotoxemia in the liver of rats. Regulation of NO production is crucial for improving the hepatic dysfunction. We have previously reported that, in cultured rat hepatocytes, a single cytokine interleukin–1β (IL–1β) stimulated a release of nitrite, an oxidation product of NO, into culture medium dose– and time–dependently. The objective of this study was to investigate an ability of the anti–inflammatory drug NaSA to affect the production of NO in hepatocytes. IL–1β increased levels of inducible NO synthase (iNOS) messenger RNA (mRNA) with a maximal effect at 8 hours in primary cultures of rat hepatocytes. Nuclear factor–κB (NF–κB), that is an important nuclear factor protein in iNOS gene transcription in response to inflammatory mediators, also appeared in the nuclear fraction of hepatocytes 1 hour after addition of IL–1β. Sodium salicylate markedly inhibited the NO formation induced by IL–1β, but did not affect NF–κB activation and iNOS mRNA induction. Western blot analysis revealed that sodium salicylate (NaSA) blocked a step of iNOS protein synthesis. These findings indicate that NaSA may reduce hepatic injury by preventing the induction of NO formation in response to IL–1β at the posttranscriptional step.

Coordination of Nuclear and Chloroplast Gene Expression in Plant Cells

Plastid proteins are encoded in two genomes, one in the nucleus and the other in the organelle. The expression of genes in these two compartments in coordinated during development and in response to environmental parameters such as light. Two converging approaches reveal features of this coordination: the biochemical analysis of proteins involved in gene expression, and the genetic analysis of mutants affected in plastid function or development. Because the majority of proteins implicated in plastid gene expression are encoded in the nucleus, regulatory processes in the nucleus and in the cytoplasm control plastid gene expression, in particular during development. Many nucleus-encoded factors involved in transcriptional and posttranscriptional steps of plastid gene expression have been characterized. We are also beginning to understand whether and how certain developmental or environmental signals perceived in one compartment may be transduced to the other.

3-Deazaadenosine analogues inhibit the production of tumour necrosis factor-alpha in RAW264.7 cells stimulated with lipopolysaccharide.

The effects of 3-deazaadenosine (DZA), 3-deaza(+/-)-aristeromycin (DZAri) and 3-deazaneplanocin (DZNep) on tumour necrosis factor-alpha (TNF-alpha) production were examined in the mouse macrophage cell line, RAW264.7, stimulated with lipopolysaccharide (LPS). The 3-deazaadenosine analogues inhibited the TNF-alpha production and the inhibition was dependent upon the concentration of the analogue. DZA reduced the level of TNF-alpha mRNA suggesting that DZA acts at a transcriptional step. In contrast, DZAri and DZNep had little effect on mRNA levels for TNF-alpha, implying that these compounds inhibit a post-transcriptional or translational biosynthetic step of TNF-alpha synthesis. The observation that homocysteine (Hcy) potentiated the DZA inhibition of TNF-alpha production and of TNF-alpha mRNA levels suggests that the inhibition of TNF-alpha production may be caused by elevated levels of 3-deazaadenosylhomocysteine (DZAHcy). The results show that the 3-deazaadenosine analogues are potent inhibitors of TNF-alpha production in the RAW264.7 cell line stimulated with LPS and suggest that these analogues may be effective agents for the treatment of diseases in which TNF-alpha plays an important pathogenic role.

Beating affects the posttranscriptional regulation of alpha-myosin mRNA in cardiac cultures

Contractile arrest of cardiac myocytes results in increased abundance of alpha-myosin heavy chain (MHC) mRNA but decreased alpha-MHC protein content. Our aim is to determine the posttranscriptional mechanisms regulating alpha-MHC mRNA-protein uncoupling in cultured neonatal rat hearts during altered contractile activity. Spontaneously contracting myocytes were arrested by the use of verapamil (10 mumol/l; a Ca(2+)-channel blocker) or by 2,3-butanedione monoxime (5 mmol/l; a cross-bridge inhibitor). Inhibition of transcription with amanitin (0.5 mumol/l) decreased the alpha-MHC mRNA in normally beating myocytes to a minimal baseline. However, the alpha-MHC mRNA did not fall this low in amanitin-treated nonbeating myocytes. Concurrently, the alpha-MHC mRNA shifted toward a heavier polysome complex when beating was blocked. Together, these data suggest that contractile arrest regulates alpha-MHC mRNA abundance posttranscriptionally by stabilizing the message at the elongation phase of translation. These posttranscriptional regulatory steps are dependent on beating itself and are independent of Ca2+ entry.

Spliced exons of adenovirus late RNAs colocalize with snRNP in a specific nuclear domain.

Posttranscriptional steps in the production of mRNA include well characterized polyadenylation and splicing reactions, but it is also necessary to understand how RNA is transported within the nucleus from the site of its transcription to the nuclear pore, where it is translocated to the cytoplasmic compartment. Determining the localization of RNA within the nucleus is an important aspect of understanding RNA production and may provide clues for investigating the trafficking of RNA within the nucleus and the mechanism for its export to the cytoplasm. We have previously shown that late phase adenovirus-infected cells contain large clusters of snRNP and non-snRNP splicing factors; the presence of these structures is correlated with high levels of viral late gene transcription. The snRNP clusters correspond to enlarged interchromatin granules present in late phase infected cells. Here we show that polyadenylated RNA and spliced tripartite leader exons from the viral major late transcription unit are present in these same late phase snRNP-containing structures. We find that the majority of the steady state viral RNA present in the nucleus is spliced at the tripartite leader exons. Tripartite leader exons are efficiently exported from the nucleus at a time when we detect their accumulation in interchromatin granule clusters. Since the enlarged interchromatin granules contain spliced and polyadenylated RNA, we suggest that viral RNA may accumulate in this late phase structure during an intranuclear step in RNA transport.

Regulation of gene expression in chloroplasts of higher plants.

Chloroplasts contain their own genetic system which has a number of prokaryotic as well as some eukaryotic features. Most chloroplast genes of higher plants are organized in clusters and are cotranscribed as polycistronic pre-RNAs which are generally processes into many shorter overlapping RNA species, each of which accumulates of steady-state RNA levels. This indicates that posttranscriptional RNA processing of primary transcripts is an important step in the control of chloroplast gene expression. Chloroplast RNA processing steps include RNA cleavage/trimming, RNA splicing, ENA editing and RNA stabilization. Several chloroplast genes are interrupted by introns and therefore require processing for gene function. In tobacco chloroplasts, 18 genes contain introns, six for tRNA genes and 12 for protein-encoding genes. A number of specific proteins and RNA factors are believed to be involved in splicing and maturation of pre-RNAs in chloroplasts. Processing enzymes and RNA-binding proteins which could be involved in posttranscriptional steps have been identified in the last several years. Our current knowledge of the regulation of gene expression in chloroplasts of higher plants is overviewed and further studies on this matter are also considered.

Post-transcriptional regulation of chloroplast gene expression in Chlamydomonas reinhardtii.

The biosynthesis of the photosynthetic apparatus depends on the concerted action of the nuclear and chloroplast genetic systems. Numerous nuclear and chloroplast mutants of Chlamydomonas deficient in photosynthetic activity have been isolated and characterized. While several of these mutations alter the genes of components of the photosynthetic complexes, a large number of the mutations affect the expression of chloroplast genes involved in photosynthesis. Most of these mutations are nuclear and only affect the expression of a single chloroplast gene. The mutations examined appear to act principally at post-transcriptional steps such as RNA stability, RNA processing, cis- and trans-splicing and translation. Directed chloroplast DNA surgery through biolistic transformation has provided a powerful tool for identifying important cis elements involved in chloroplast gene expression. Insertion of chimeric genes consisting of chloroplast regulatory regions fused to reporter genes into the chloroplast genome has led to the identification of target sites of the nuclear-encoded functions affected in some of the mutants. Biochemical studies have identified a set of RNA-binding proteins that interact with the 5'-untranslated regions of plastid mRNAs. The binding activity of some of these factors appears to be modulated by light and by the growth conditions.

Optimizing expression of transgenes with an emphasis on post-transcriptional events.

Introducing a foreign gene into a new plant host does not always result in a high level of expression of the incoming gene. Numerous promoters have been used to express foreign genes in different plant tissues, but there are sometime various features of the new gene which are deleterious to expression in the new host. There are a number of post-transcriptional steps in the expression of a gene and sometimes sequences present in a particular coding region can resemble the signals which initiate these processing steps. When aberrantly carried out, these steps diminish the level of expression. By removing such fortuitous signals, one can dramatically increase expression of a transgene in plants. Ensuring proper protein folding and/or targeting the protein product to a particular cellular compartment can also be used to increase the level of protein obtained. The various methods used to optimize expression of a foreign gene in plants by concentrating on post-transcriptional events are discussed.

Posttranscriptional Regulation of Glucose-6-phosphate Dehydrogenase by Dietary Polyunsaturated Fat

The activity of glucose-6-phosphate dehydrogenase (G6PD) is inhibited by the addition of polyunsaturated fat (PUFA) to a high carbohydrate diet. To define the regulated step, we measured enzyme activity, accumulation of G6PD mRNA, and transcriptional activity of the gene. At steady-state, G6PD activity and mRNA abundance were inhibited by 80% in the livers of mice fed a high-fat diet (6% safflower oil) compared to mice fed a low-fat diet (1% safflower oil). Inhibition of mRNA accumulation was 20% by 4 h and was maximal by 9 h after beginning the high-fat diet. Changes in mRNA accumulation preceded changes in enzyme activity, indicating pretranslational regulation. The rapid kinetics of G6PD mRNA accumulation depended on prior dietary history of the mice. In meal-trained mice, abundance of G6PD mRNA increased by twofold within 4 h of the onset of a low-fat meal and was maximal by 12 h. In contrast, an increase in G6PD mRNA was not observed until 12 h after refeeding starved mice and the increase was maximal (12-fold) by 27 h. Transcriptional activity of the gene was measured using the nuclear run-on assay. The G6PD probes were rigorously screened for repetitive elements and for transcription of the noncoding strand of the gene. Throughout the time course of changes in G6PD mRNA accumulation due to PUFA or refeeding, transcriptional activity of the gene did not change. Therefore, regulation of G6PD expression by nutritional status occurs at a posttranscriptional step.

Regulation of the yeast vacuolar aminopeptidase Y gene (APY1) expression.

The steady-state levels of the aminopeptidase Y (APY1) transcript and the level of assayable aminopeptidase Y activity were measured under a variety of nutritional conditions. Specific APY1 mRNA is less abundant in cells grown in minimal medium than in cells grown in rich medium, while active enzyme levels follow just the opposite pattern. Aminopeptidase Y activity decreases when cells are deprived of glucose without a concomitant fall in mRNA levels. Production of aminopeptidase Y is not markedly affected by nitrogen regulation. APY1 gene expression is not disturbed in heat-shocked cells. Our data support the idea that the main control event in vacuolar aminopeptidase Y expression is a post-transcriptional step.

Regulation of the yeast vacuolar aminopeptidase Y gene ( APY1) expression

The steady-state levels of the aminopeptidase Y ( APY1) transcript and the levels of assayable aminopeptidase Y activity were measured under a variety of nutritional conditions. Specific APY1 mRNA is less abundant in cells grown in minimal medium than in cells grown in rich medium, while active enzyme levels follow just the opposite pattern. Aminopeptidase Y activity decreases when cells are deprived of glucose without a concomitant fall in mRNA levels. Production of aminopeptidase Y is not markedly affected by nitrogen regulation. APY1 gene expression is not disturbed in heat-shocked cells. Our data support the idea that the main control event in vacuolar aminopeptidase Y expression is a post-transcriptional step.

Differential expression of alpha- and beta-enolase genes during rat heart development and hypertrophy

We have analyzed the transition between isoforms of the glycolytic enzyme enolase (2-phospho-D-glycerate hydrolyase; EC 4.2.1.11) in rat heart during normal and pathological growth. A striking fall in embryonic alpha-enolase gene expression occurs during cardiac development, mostly controlled at pretranslational steps. In fetal and neonatal hearts, muscle-specific beta-enolase gene expression is a minor contributor to total enolase. Control mechanisms of beta-enolase gene expression must include posttranscriptional steps. Aortic stenosis induces a rapid and drastic decrease in beta-enolase transcript level in cardiomyocytes, followed by the fall in beta-subunit level. In contrast, alpha-enolase transcript level is not significantly altered, although the corresponding subunit level increases in nonmuscle cells. We conclude that, like fetal heart, hypertrophic heart is characterized by a high ratio of alpha- to beta-enolase subunit concentrations. This study indicates that the decrease in beta-enolase gene expression may be linked to beneficial energetic changes in contractile properties occurring during cardiac hypertrophy.

Regulation of DNA methyltransferase during differentiation of F9 mouse embryonal carcinoma cells

DNA becomes demethylated when F9 mouse embryonal carcinoma cells differentiate into parietal endoderm. DNA methyltransferase (DNA-MTase) activity decreased by 50% during 1 week of differentiation. The level of DNA-MTase mRNA was also diminished accordingly, but the transcription rate of the DNA-MTase gene measured by run-on transcription was essentially unchanged, indicating regulation of DNA-MTase expression at a posttranscriptional step. The decline of DNA-MTase mRNA paralleled that of histone H3 mRNA in accord with the notion that DNA-MTase is preferentially expressed in the S phase of the cell cycle. Since DNA-MTase expression decreases in parallel with DNA synthesis, DNA demethylation during differentiation of F9 cells appears not to be due to limited expression of DNA-MTase. However, the plasmid pAFP7000CAT, alpha-fetoprotein (AFP), which is strongly de novo methylated when transfected into F9 stem cells became only weakly methylated after transfection into the F9 parietal endoderm derivative P1, indicating that the activity of DNA-MTase within parietal endoderm cells is more strongly diminished than is apparent from measurements of mRNA amounts and of overall DNA-MTase activity in vitro. The discrepancy between DNA-MTase expression and its actual activity within the cell indicates the existence of a novel mechanism controlling the activity of DNA-MTase.

Post-transcriptional regulation of gene expression in hippocampal neurons by glutamate receptor activation

Previous work from this laboratory has documented that glutamate receptor activation and extracellular calcium entry into hippocampal neurons caused a long-lasting down-regulation of ligatin mRNA and protein. Here, we investigated whether glutamate reduced ligatin mRNA levels by decreasing the transcriptional activity of the gene and/or by regulating post-transcriptional RNA processing steps including mRNA stability. Using nuclear run-on assays, it was demonstrated that transcriptional activity of the ligatin gene was not significantly decreased after glutamate receptor activation. Further, Northern analysis of RNA from neurons maintained in the presence of the transcription inhibitor, α-amanitin, showed that glutamate shortened the half life of the ligatin message from 10 h to 58 min. This post-transcriptional destabilization of ligatin mRNA was mimicked by NMDA, dependent on Ca 2+, blocked by MK801, and not affected by AMPA and kainic acid, indicating that message stability was governed by changes in intracellular calcium. Moreover, using in situ hybridization and confocal microscopy, we showed that glutamate and NMDA decreased ligatin message within dendritic and somal regions without increasing nuclear levels. These findings demonstrated that glutamate receptor activation altered neuronal gene expression posttranscriptionally by destabilizing mRNA. Our data suggest that post-transcriptional regulation of gene expression may be part of the normal receptor-mediated regulatory program of plasticity and provides the first description of a glutamate receptor-modulated, calcium-dependent mechanism which rapidly destabilizes mRNA in neurons.

Azalanstat (RS-21607), a lanosterol 14α-demethylase inhibitor with cholesterol-lowering activity

Agents that inhibit hepatic cholesterol biosynthesis reduce circulating cholesterol levels in experimental animals and humans, and may be of pharmacological importance in the prevention of atherosclerosis. Azalanstat (RS-21607), a synthetic imidazole, has been shown to inhibit cholesterol synthesis in HepG2 cells, human fibroblasts, hamster hepatocytes and hamster liver, by inhibiting the cytochrome P450 enzyme lanosterol 14α-demethyiase. When administered orally to hamsters fed regular chow, RS-21607 (50 mg/kg/day) lowered serum cholesterol in a dose-dependent manner (ED 50 = 62 mg/kg) in a period of 1 week. It preferentially lowered low density lipoprotein (LDL) cholesterol and apo B relative to high density lipoprotein (HDL) cholesterol and apo A-1. It also lowered plasma cholesterol levels in hamsters fed a high saturated fat and cholesterol diet. RS-21607 inhibited hepatic microsomal hydroxymethylglutaryl-CoA (HMG-CoA) reductase activity in hamsters in a dosedependent manner (ED 50 = 31 mg/kg), and this was highly correlated with serum cholesterol lowering ( r = 0.97). Cholesterol lowering by azalanstat and cholestyramine was additive, and the increase in HMG-CoA reductase brought about by cholestyramine was attenuated significantly by azalanstat. In vitro studies with HepG2 cells indicated that this modulation of reductase activity was indirect, occurring at a post-transcriptional step, and it is proposed that a regulatory oxysterol derived from dihydrolanosterol (or lanosterol) may be responsible for this regulation. Azalanstat does not appear to lower circulating cholesterol in the hamster by up-regulation of the hepatic LDL receptor, suggesting that other mechanisms are involved. Orally administered azalanstat (50–75 mg/kg) stimulated hepatic microsomal cholesterol 7α-hydroxylase activity by 50–400% in hamsters, and it is postulated that this may result from modified cholesterol absorption and bile acid synthesis.

Short- and long-term mechanisms of tau regulation in PC12 cells.

Induction by nerve growth factor of neurite outgrowth in PC12 cells is transcription-dependent and is associated with the accumulation of tau protein. It was recently shown that short-term treatment with staurosporine, a protein kinase alkaloid inhibitor, induced an elevation of tau protein levels and outgrowth of stable neurites. In this study, we analyzed the mechanism(s) by which nerve growth factor and staurosporine exert their effects on tau levels. We demonstrate that nerve growth factor affects tau mRNA stability, thus contributing to the observed increase in tau mRNA levels. On the other hand, tau mRNA levels were not affected by the treatment with staurosporine. We also demonstrate that the phosphorylation of tau protein was reduced after treatment of PC12 cells with nerve growth factor or staurosporine, as shown by immunoblot analysis using specific antibodies and alkaline phosphatase treatment. Thus, regulation of tau levels by nerve growth factor appears to be mediated by transcriptional, post-transcriptional and posttranslational steps, whereas the effect of staurosporine on tau levels may be attributed to its effect on the state of phosphorylation of the protein.

CT-2576, an inhibitor of phospholipid signaling, suppresses constitutive and induced expression of human immunodeficiency virus.

Viruses such as human immunodeficiency virus (HIV) require cellular activation for expression. Cellular activation in lymphoid cells is associated with augmented accumulation of certain phosphatidic acid (PA) species derived from the hydrolysis of glycan phosphatidylinositol (GPI). This suggests that activation of a phospholipid pathway may play a role in initiation of viral replication. To test this hypothesis, we examined the effect of tat gene expression on the production of cellular PA species, as the Tat protein is essential for HIV expression and has been implicated in activating the expression of multiple host cellular genes. Expression of tat increased the expression of PA. We then tested whether synthetic inhibitors of PA metabolism would inhibit activation of the HIV long terminal repeat by Tat and tumor necrosis factor alpha (TNF-alpha). CT-2576 suppressed both PA generation induced by Tat and HIV long terminal repeat-directed gene expression in response to Tat or TNF-alpha at a posttranscriptional step. CT-2576 also inhibited constitutive as well as TNF-alpha- and interleukin 6-induced expression of HIV p24 antigen in chronically infected U1 cells and in peripheral blood lymphocytes acutely infected with a clinical isolate of HIV. Pharmacological inhibition of synthesis of selected PA species may therefore provide a therapeutic approach to suppression of HIV replication.

Human T cell leukaemia virus type I env gene-transfected HeLa cells display a decrease in cell fusion ability.

The envelope (env) gene of human T cell leukaemia virus type I (HTLV-I) was inserted into an expression vector, referred to as phMTenv, under the transcriptional control of the human metallothionein IIa gene promoter (hMT-IIa). When this vector was transiently transfected in HeLa cells treated with hMT-IIa inducers, formation of multinucleated cells was observed, indicating the expression of functional surface and transmembrane glycoproteins. Of several HeLa cell clones transfected with phMTenv together with a plasmid carrying the neomycin resistance gene and isolated after selection in G418-containing medium, env mRNA was detected in only two, in the presence of hMT-IIa inducers. Viral glycoproteins were found to be weakly expressed as detected in immunoprecipitation assays of 125I-surface-labelled cells. These env-transfected HeLa cell clones, although unable to form syncytia when cocultivated with untransfected control HeLa cells, retained the capacity to fuse with HTLV-I-producing C91PL T cells. However, a significant decrease in their fusogenic ability was observed, after treatment with hMT-IIa inducers. Under identical experimental conditions, control HeLa cell clones stably transformed with the same plasmid, but lacking the env gene, were still able to fuse with C91PL cells. These observations suggest that a post-transcriptional step in HTLV-I env expression is impaired, probably leading to the establishment of superinfection interference.