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PubMed1997

Mechanistic modeling of prokaryotic mRNA decay.

A mechanistic model of gene expression was developed to test three prevailing and sliding prokaryotic mRNA decay theories: ribosome protection of mRNA from endonucleases, 5' binding and sliding of endonucleases on mRNA, and hybrid 5' binding/ribosome protection. The discrete event simulation incorporates the molecular events that determine both cellular mRNA and protein levels. A Monte Carlo technique was used to approximate the inherent randomness of the molecular processes involved in gene expression. Each of the decay theories was tested for the ability to predict the effects of ribosome loading and translation rate on mRNA stability as well as the observed 5' to 3' directionality of mRNA decay. The modeling results show that the hybrid decay mechanism best predicts the experimentally-observed mRNA decay behaviors. The 5' binding mechanism fails to adequately predict the sensitivity of mRNA stability to changes in translation rate and ribosome loading, while the ribosome protection mechanism does not correctly predict 5' to 3' decay directionality. In addition to discriminating between the three decay theories, the simulations provide insights into hybrid decay mechanism specific details such as RNase binding and cleavage characteristics. Finally, we discuss the application of the current mechanistic model for analysing and predicting expression from more complex genetic systems.

Computer Simulation↗
PubMed2003

Interplay between transcriptional and post-transcriptional regulation of Cyp2a5 expression.

The cytochrome P450 (Cyp) 2a5 gene can be upregulated transcriptionally or by mRNA stabilization. The heterogeneous nuclear ribonucleoprotein (hnRNP) A1 interacting with the CYP2A5 mRNA has been shown to be a key post-transcriptional regulator of the Cyp2a5 gene. The aim of this study was to investigate if the transcriptional and post-transcriptional steps of Cyp2a5 expression are linked. This was done by modifying the transcription rate with transcriptional inducers (phenobarbital and cyclic AMP) and inhibitors (actinomycin D and 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole) and analyzing the effects upon post-transcriptional events. We found that inhibition of transcription led to relocalization of hnRNP A1 from the nucleus to the cytoplasm, to its strongly increased binding to the cytoplasmic CYP2A5 mRNA and to CYP2A5 mRNA stabilization. In contrast, stimulated transcription resulted in increased binding of nuclear hnRNP A1 to the Cyp2a5 promoter, and overexpression of hnRNP A1 led to stimulated transcription of a Cyp2a5 promoter-driven luciferase recombinant. This strongly suggests that the transcriptional and post-transcriptional stages of Cyp2a5 expression are interrelated and that the nucleocytoplasmic shuttling hnRNP A1 may coordinate these different steps.

Animals↗
PubMed1997

The spinach chloroplast endoribonuclease CSP41 cleaves the 3'-untranslated region of petD mRNA primarily within its terminal stem-loop structure.

3'-Untranslated region stem-loop structures are major determinants of chloroplast mRNA stability. The 3' stem-loop region of spinach petD precursor mRNA (pre-mRNA), a chloroplast gene encoding subunit IV of the cytochrome b6.f complex, forms a stable RNA-protein complex in vitro with chloroplast stem-loop binding proteins (CSPs) of 55, 41, and 29 kDa. We have previously purified CSP41 and cloned the corresponding cDNA. In vitro studies demonstrated that CSP41 is a bifunctional protein that displays both endoribonuclease and RNA-binding activities. In this work, the RNase activity of CSP41 is further characterized using the bacterially expressed protein. Our data show that CSP41 cleaves both single-stranded and double-stranded RNAs but not DNA. However, it exhibits a preference for stem-loop-containing RNAs. When the 3'-untranslated region of petD pre-mRNA is provided as a substrate, CSP41 specifically cleaves it within the stem-loop region, implying that CSP41 has an important role in the control of petD mRNA stability. Our data also show that the sequence-specific RNA-binding activity of CSP41 affects the rate, but not the specificity, of its RNase activity, suggesting that CSP41 is probably involved in other events of chloroplast RNA metabolism in addition to RNA degradation. By analyzing C-terminal deletions of CSP41, the RNase domain was located between amino acid residues 73 and 191.

Amino Acid Sequence↗
PubMed2003

AP-1 is involved in UTP-induced osteopontin expression in arterial smooth muscle cells.

Osteopontin (OPN), an RGD-containing extracellular matrix protein, is associated with arterial smooth muscle cell (SMC) activation in vitro and in vivo. Many cytokines and growth factors involved in vessel wall remodeling induce OPN overexpression. Moreover, we recently demonstrated that the extracellular nucleotide UTP also induces OPN expression and that OPN is essential for UTP-mediated SMC migration. Thus, we set out to investigate the mechanisms of OPN expression. The aim of this study was to identify transcription factors involved in the regulation of OPN expression in SMCs. First, we explored the contribution of mRNA stabilization and transcription in the increase of UTP-induced OPN mRNA levels. We show that UTP induced OPN mRNA increases via both OPN mRNA stabilization and OPN promoter activation. Then, to identify transcription factors involved in UTP-induced OPN transcription, we located a promoter element activated by UTP within the rat OPN promoter using a gene reporter assay strategy. The -96 to +1 region mediated UTP-induced OPN overexpression (+276+/-60%). Sequence analysis of this region revealed a potential site for AP-1 located at -76. When this AP-1 site was deleted, UTP-induced activation of the -96 to +1 region was totally inhibited. Thus, this AP-1 (-76) site is involved in UTP-induced OPN transcription. A supershift assay revealed that both c-Fos and c-Jun bind to this AP-1 site. Finally, we demonstrate that angiotensin II and platelet-derived growth factor, two main factors involved in vessel wall pathology, also modulated OPN expression via AP-1 activation.

Angiotensin II↗
PubMed2004

Distinct regulation of cyclooxygenase-2 by interleukin-1beta in normal and endometriotic stromal cells.

Aberrant production of cyclooxygenase-2 (COX-2) plays pivotal roles in many pathological processes including tumorigenesis and endometriosis, although the underlying mechanism remains obscure. Herein we report evidence to demonstrate that COX-2 is distinctly regulated by IL-1beta in normal and endometriotic stroma. Ectopic endometriotic stromal cell is at least 100 times more sensitive to IL-1beta treatment, compared with its eutopic counterpart. Induction of COX-2 expression in normal endometrial stroma by IL-1beta is primary due to enhancement of COX-2 mRNA stability. In contrast, IL-1beta not only increases COX-2 mRNA stability but also up-regulates COX-2 promoter activity in ectopic endometriotic stroma. Induction of COX-2 promoter activity by IL-1beta is mediated via MAPK-dependent phosphorylation of cAMP-responding element binding protein. Promoter activity and EMSAs demonstrate that a cAMP response element site located at -571/-564 of COX-2 promoter is critical for IL-1beta-induced COX-2 gene expression. Our results indicate that elevation of COX-2 expression in endometriotic tissues may result from increased sensitivity to proinflammatory cytokines such as IL-1beta, which is consistently present in the peritoneal fluid of endometriosis patients. Distinct regulation of COX-2 gene by IL-1beta may play a critical role in pathophysiological processes such as cancer formation and endometriosis.

Cells, Cultured↗
PubMed1995

The intracellular IL-1 receptor antagonist alters IL-1-inducible gene expression without blocking exogenous signaling by IL-1 beta.

The epithelium-associated tissue distribution of the intracellular IL-1R antagonist (icIL-1Ra) suggests that it functions as a novel regulatory molecule for IL-1 in somatic tissues. We examined the role of the icIL-1Ra in IL-1 beta-induced responses in human ovarian cancer cells because ovarian surface epithelium expresses transcripts for the icIL-1Ra, and the majority of ovarian cancers arise from these cells. Several human ovarian and cervical cancer cell lines spontaneously express the icIL-1Ra. icIL-1Ra-expressing cells did not have altered growth characteristics or altered short term responses to IL-1 compared with icIL-1Ra-nonexpressing cells. While a 90-min exposure to IL-1 beta resulted in increased steady state cytokine mRNA levels in all cells, icIL-1Ra-positive cells were incapable of maintaining IL-1-beta-induced expression of GRO mRNA. This did not result from decreased transcriptional activity of the GRO gene, but reflected differences in mRNA stability and/or degradation. To determine whether the icIL-1Ra altered mRNA stability, we used a retroviral expression vector to express the icIL-1Ra in an icIL-1Ra-negative cell line. The resulting cells displayed a profile of IL-1 beta-induced genes analogous to that found in cells spontaneously expressing icIL-1Ra. These data show for the first time an intrinsic biologic activity for the icIL-1Ra. The capacity to selectively alter IL-1-induced gene expression suggests that this version of the IL-1Ra is a unique intracellular inhibitor that attenuates IL-1 responses at a point downstream of the initial IL-1/IL-1 receptor interaction.

Female↗
PubMed1994

Agonist and cyclic AMP-mediated regulation of beta 1-adrenergic receptor mRNA and gene transcription in rat C6 glioma cells.

Exposure of rat C6 glioma cells to either agonists or agents that increase cyclic AMP levels leads to down-regulation of beta 1-adrenergic receptors (beta 1 AR) as measured by loss of radioligand binding sites. The present study examines the influence of isoproterenol and forskolin treatment on levels of beta 1 AR mRNA, mRNA stability, and gene transcription rate. Isoproterenol treatment of C6 cells altered beta 1 AR mRNA levels in a biphasic manner; i.e., short-term exposure (30-60 min) increased by 50%, whereas longer exposure (2-6 h) decreased by 50% the levels of beta 1 AR mRNA. The extent of both the up- and down-regulation was dependent on agonist concentration. Similar regulation of beta 1 AR mRNA was observed in forskolin-treated cells. Pretreatment of the cells with Pseudomonas exotoxin A, a potent inhibitor of protein synthesis, completely blocked isoproterenol- and forskolin-mediated down-regulation of beta 1 AR mRNA, and thereby potentiated the increase in receptor mRNA up to fourfold over the 6-h time course. The mechanisms underlying beta 1 AR mRNA down-regulation were examined. The half-life of beta 1 AR mRNA was slightly increased (from 61 to 77 min) after a 2-h exposure of the cells to either isoproterenol or forskolin. Nuclear run-on analysis demonstrated that the rate of beta 1 AR gene transcription was increased after isoproterenol incubation for 60 min, but then decreased after 90-240 min, consistent with the time course for up- and down-regulation of beta 1 AR mRNA. Isoproterenol treatment (120 min) also decreased the level of beta 1 AR nascent transcripts, purified by affinity chromatography of RNA isolated from 4-thiouridine-pulsed cells. The results demonstrate that beta 1 AR mRNA has a relatively short half-life and that agonist regulation of beta 1 AR mRNA is mediated by activation of the cyclic AMP system. Moreover, the results indicate that agonist regulation of beta 1 AR mRNA occurs at the level of beta 1 AR gene transcription, not mRNA stability. Finally, the observed requirement for protein synthesis indicates that beta 1 AR mRNA down-regulation may be mediated by the induction of a repressor of the beta 1 AR gene.

Adrenergic beta-1 Receptor Agonists↗
PubMed2003

Characterization of adapt33, a stress-inducible riboregulator.

We have identified adapt33 as a multiple stress-responsive gene that is induced under conditions of a cytoprotective "adaptive response." adapt33 RNA does not contain any appreciable open reading frame nor produce a protein product and is therefore classified as a stress-inducible riboregulator. Although a number of oxidant stress-modulated, protein-encoding genes have been reported and characterized, very few stress-inducible riboregulator RNAs are known. Here we extend previous studies toward understanding the underlying regulation of expression and function of this rare mammalian riboregulator. mRNA stability and transcription studies determined that adapt33 induction by hydrogen peroxide is at the mRNA stability level, and that adapt33 has a very short half-life. Surprisingly, adapt33 mRNA also exhibits altered electrophoretic migration in response to both hydrogen peroxide and cis-platinum treatment. Although no transcriptional modulation in response to hydrogen peroxide was observed, fusion promoter constructs revealed that adapt33 has an unusually strong promoter that is active in both hamster and human cells. Analysis of expression following the stimulation of apoptosis with hydrogen peroxide and staurosporine revealed a strong correlation with apoptosis, suggesting a possible novel, noncoding RNA component of the apoptotic mechanism. We conclude that adapt33 is a stress-inducible, apoptosis-associated RNA with unique structural and gene promoter characteristics.

Animals↗
PubMed1992

Transcriptional and posttranscriptional regulation of the proopiomelanocortin gene in the pars intermedia of the pituitary gland of Xenopus laevis.

In the melanotrope cells of the intermediate pituitary gland of the amphibian Xenopus laevis, expression of the POMC gene is under physiological control, i.e. it is 20- to 30-fold higher in animals adapted to a black background compared to animals adapted to a white background. To investigate whether changes in POMC messenger RNA stability contribute to this difference in expression, a steady state kinetic model for mRNA degradation was used to determine the half-life of POMC mRNA during the induction and deinduction of POMC gene expression in melanotrope cells. During induction of the POMC gene the half-life of POMC mRNA was 3- to 4-fold longer than during deinduction. This difference in mRNA stability is, however, not sufficient to account for the 20- to 30-fold difference in the steady state levels of POMC mRNA between the two physiological conditions. Results from experiments with the protein synthesis inhibitor cycloheximide and the mRNA synthesis inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzidimidazole suggest 1) that during induction of POMC gene expression no de novo protein synthesis is required and, 2) that deinduction of POMC gene expression requires transcriptional activation of an mRNA-degradation system. Pulse-labeling experiments with [3H]uridine showed that in neurointermediate lobes of white-adapted animals there is an 8-fold higher amount of newly synthesized POMC mRNA than in lobes of black animals. This suggests a fast in vitro induction of POMC gene transcription. The dopamine D2-receptor agonist apomorphine decreased POMC mRNA biosynthesis about 5-fold, which confirms that regulation of POMC gene expression includes a transcriptional component. In conclusion, our results demonstrate that during the physiological process of background adaptation the regulation of POMC gene expression in Xenopus melanotrope cells is exerted by alterations in POMC gene transcription as well as in POMC mRNA stability.

Animals↗
PubMed1999

Na(+)-K(+)-ATPase gene regulation by glucocorticoids in a fetal lung epithelial cell line.

The Na(+) pump, Na(+)-K(+)-ATPase, along with the Na(+) channel is essential for the removal of alveolar solute and fluid perinatally. Because Na(+)-pump mRNA and activity increase before birth and maternal glucocorticoids (GCs) influence Na(+)-K(+)-ATPase mRNA expression in fetal rat lung, we hypothesized that GCs increased Na(+)-K(+)-ATPase gene expression in a fetal lung epithelial cell line. After 24 h of exposure, dexamethasone increased the steady-state levels of Na(+)-K(+)-ATPase alpha(1) and beta(1) mRNA in a fetal rat lung epithelial cell line in a dose-dependent fashion (10(-7) to 10(-5) M). The maximal increase in mRNA levels was 3. 8-fold for alpha(1) and 2.8-fold for beta(1). The increase in mRNA was detected as early as 6 h for the beta(1)-subunit and 18 h for the alpha(1)-subunit, and both peaked at 24 h. This gene upregulation was not due to increased mRNA stability based on mRNA half-life determination after actinomycin D inhibition. Transfection experiments with alpha(1) and beta(1) promoter-reporter constructs demonstrated 3.2 +/- 0.5- and 2.6 +/- 0.4-fold increases, respectively, in promoter activity, consistent with transcriptional activation of the promoter-reporter construct. These findings, increased promoter activity with no change in stability, indicate that GCs increased Na(+)-K(+)-ATPase transcription in a fetal lung epithelial cell line.

Animals↗
PubMed2004

Oxysterols induce cyclooxygenase-2 expression in cholangiocytes: implications for biliary tract carcinogenesis.

Cyclooxygenase-2 (COX-2), which is expressed by cholangiocytes in biliary tract disorders, has recently been implicated in biliary tract carcinogenesis. The mechanisms responsible for this COX-2 expression remain unclear. In human diseases, bile contains oxygenated derivatives of cholesterol (oxysterols) which possess diverse biological properties. Therefore, we determined if oxysterols modulate COX-2 expression. The effect of an oxysterol (22(R)-hydroxycholesterol, 22-HC) on COX-2 expression in KMBC cells, a human cholangiocarcinoma cell line, was examined. 22-HC enhanced COX-2 protein expression. This oxysterol activated p42/44 and p38 MAPK, but not JNK 1/2. A p42/44 MAPK inhibitor did not block COX-2 induction, while p38 MAPK inhibitor effectively attenuated COX-2 induction. Although COX-2 mRNA levels were increased by 22-HC, this increase was not transcriptionally regulated, as 22-OH did not increase activity in a COX-2 promoter gene assay. In contrast, COX-2 mRNA stability was augmented by 22-HC treatment, and this effect was reversed by a p38 MAPK inhibitor. In conclusion, the results demonstrate that the oxysterol 22-HC stabilizes COX-2 mRNA via a p38 MAPK-dependent mechanism. This enhanced COX-2 protein expression by oxysterols may participate in the genesis and progression of cholangiocarcinoma.

Bile Duct Neoplasms↗
PubMed1998

Ethanol and regulation of the NMDA receptor subunits in fetal cortical neurons.

Previous studies have shown that chronic ethanol treatment up-regulates the expression of the N-methyl-D-aspartate (NMDA) receptor number and function both in vitro and in vivo. In vitro chronic ethanol treatment specifically augments mRNA levels of the R2B subunit without altering R1 subunit mRNA levels, although similar treatment results in increased levels of both R1 and R2B polypeptides. To further delineate the molecular mechanisms involved in differential regulation of NMDA receptor subunits by chronic ethanol treatment (50 mM, 5 days), we have determined the mRNA stability of the NMDA R1 and R2B subunits and the transcription rate of the respective genes using mouse fetal cortical neurons. Our observations demonstrated that ethanol stabilized the NMDA R1 mRNA over the time period examined (24 h) without altering the stability of the R2B mRNA. Chronic exposure of fetal cortical neurons to ethanol enhanced the rate of R2B gene transcription approximately twofold. Taken together, these results suggest that up-regulation of the NMDA receptor number seen in cultured cortical neurons after chronic ethanol treatment is due to the regulation of the NMDA R2B receptor subunit at the transcriptional level and that of the NMDA R1 subunit mainly at the posttranscriptional level.

Animals↗
PubMed2005

NF-kappaB inhibitors stabilize the mRNA of high-affinity type-2 cationic amino acid transporter in LPS-stimulated rat liver.

BACKGROUND: Induction of inducible nitric oxide synthase (iNOS) results in nitric oxide (NO) overproduction during endotoxemia. Cellular uptake of L-arginine, modulated by the isozymes of type-2 cationic amino acid transporters (CAT), including CAT-2, CAT-2A and CAT-2B, has been reported to be a crucial factor in the regulation of iNOS activity. We sought to elucidate the expression of CAT-2 isozymes and the role of nuclear factor-kappaB (NF-kappaB) in this expression in lipopolysaccharide (LPS)-treated rat liver. METHODS: Adult male Sprague-Dawley rats were randomly given intravenous (i.v.) injections of normal saline (N/S), LPS, LPS preceded by an NF-kappaB inhibitor (PDTC, dexamethasone or salicylate) or an NF-kappaB inhibitor alone. After injection, rats were sacrificed at different times and enzyme expression and liver injury were examined. Hepatic and systemic NO production were also measured. RESULTS: CAT-2, CAT-2A and CAT-2B were constitutively expressed in un-stimulated rat liver. LPS stimulation not only significantly increased iNOS mRNA and NO concentrations but also decreased the mRNA concentrations of CAT-2 and CAT-2B, but not CAT-2A, in a time-dependent manner. LPS-induced hepatic and systemic NO overproduction was associated with hepatocellular injury. Pre-treatment with NF-kappaB inhibitors significantly attenuated LPS-induced iNOS induction as well as CAT-2/CAT-2B mRNA destabilization, which was associated with significant inhibition of NO biosynthesis and less liver injury. CONCLUSION: NF-kappaB inhibitors stabilize CAT-2 and CAT-2B mRNA in LPS-stimulated rat liver. The hepatic CAT-2/CAT-2B pathway may be a constitutive part of cytoprotective mechanisms against sepsis.

Alanine Transaminase↗
PubMed2002

Multiple control of interleukin-8 gene expression.

Interleukin (IL)-8, a prototypic human chemokine, was detected more than a decade ago as the founding member of the chemokine superfamily. One of the most remarkable properties of IL-8 is the variation of its expression levels. In healthy tissues, IL-8 is barely detectable, but it is rapidly induced by ten- to 100-fold in response to proinflammatory cytokines such as tumor necrosis factor or IL-1, bacterial or viral products, and cellular stress. Recently, significant advances in the understanding of signaling pathways, which coordinately regulate IL-8 transcription as well as mRNA stabilization in response to external stimuli, have been made. Maximal IL-8 amounts are generated by a combination of three different mechanisms: first, derepression of the gene promoter; second, transcriptional activation of the gene by nuclear factor-kappaB and JUN-N-terminal protein kinase pathways; and third, stabilization of the mRNA by the p38 mitogen-activated protein kinase pathway. In that way, cells are able to rapidly increase and at the same time, to fine-tune the amount of IL-8 secreted and thereby control the extent of leukocytes attracted to sites of tissue injury.

Animals↗
PubMed1996

Multiple regions of the Arabidopsis SAUR-AC1 gene control transcript abundance: the 3' untranslated region functions as an mRNA instability determinant.

The small-auxin-up-RNA (SAUR) transcripts are rapidly induced by auxin and are among the most short-lived mRNAs in higher plants. In this study, we investigate the regulation of SAUR-AC1, a well characterized SAUR gene of Arabidopsis. Be examining the expression of chimeric genes in transgenic tobacco, we demonstrate that the promoter region of SAUR-AC1 mediates auxin induction. Sequences downstream of the promoter region were found to limit mRNA accumulation in a manner that was independent of auxin treatment. Both the coding region and the 3' untranslated region (UTR) of SAUR-AC1 independently contribute to this limitation. Effects on mRNA stability were assayed using chimeric genes under the control of the tetracycline-repressible Top10 promoter. mRNA half-life analysis following tetracycline treatment showed that the SAUR-AC1 coding region does not contain elements that decrease mRNA stability. In contrast, the 3' UTR was found to act as a potent mRNA instability determinant. This finding and the general utility of the Top10 system should provide the means to elucidate mRNA decay pathways that are potentially novel and specific for certain unstable transcripts.

Arabidopsis↗
PubMed1990

Stability of IL-2 mRNA in T lymphocytes is controlled by a protein kinase C-regulated mechanism.

The rate of the degradation of interleukin 2 (IL-2) mRNA produced in stimulated human tonsillar lymphocytes was found to be significantly decreased in cells continuously stimulated with a calcium ionophore, A23187, and a phorbol ester, phorbol 12, 13-dibutylate (PDB) as compared with that in unstimulated cells. When the lymphocytes were stimulated with A23187 and PDB, IL-2 mRNA reached a maximum level at 8 h and gradually decreased to almost the base line by 27 h. IL-2 mRNA produced was rapidly degraded when the stimulants were washed out at 12 h and the cells further cultured in the presence of actinomycin D, which stops mRNA synthesis. However, the stability of IL-2 mRNA was increased by the addition of PDB or A23187. A maximal effect was observed when both were added. The effect of PDB was dose-dependent and inhibited by the inhibitors of protein kinase C (PKC), staurosporine, and K252a, suggesting the involvement of PKC in the control of IL-2 mRNA stability. The involvement of protein phosphorylation in the regulating mechanism of IL-2 mRNA stability was supported by the fact that the addition of okadaic acid, which inhibits serine/threonine protein phosphatases, resulted in an increase in the stability of IL-2 mRNA. Further study demonstrated that the rate of degradation of 32P-labeled IL-2 mRNA, which was prepared by cell-free transcription of IL-2 cDNA, in the polysomal fraction obtained from PDB-stimulated lymphocytes was decreased compared with that obtained from unstimulated lymphocytes. These results indicate the presence of a mechanism controlling the stability of IL-2 mRNA that is regulated by PKC.

Calcimycin↗
PubMed1994

Identification and characterization of an antisense RNA transcript (gfg) from the human basic fibroblast growth factor gene.

Basic fibroblast growth factor (bFGF) is an autocrine growth factor that is overexpressed in glial tumor cells and promotes their unregulated proliferation. We have previously reported that increased messenger RNA (mRNA) stability contributes to the elevated steady state levels of bFGF mRNA in human U87-MG glioma cells. Stability of bFGF mRNA is regulated by a natural antisense transcript in Xenopus oocytes, but the mammalian equivalent of this transcript has not previously been described. We were interested in identifying the human equivalent of this antisense transcript in order to study its role in bFGF mRNA stability. Analysis of the 3'-untranslated region of the 6.7-kilobase human bFGF mRNA revealed two areas of greater than 75% homology to exons 3 and 4 of the Xenopus antisense transcript, separated by 4300 basepairs of nonhomologous sequence. We used reverse transcription-polymerase chain reaction to amplify, clone, and sequence a 301-basepair fragment of the antisense splice variant from U87-MG cells. The clone (gfg-1) is 73% identical to the Xenopus sequence, with a conserved splice junction and an open reading frame. Strand-specific gfg-1 complementary RNA probes detect a 1.5-kilobase mRNA transcript in normal rat tissues and human T47D breast cancer cells, which contain very low levels of bFGF mRNA. In contrast, antisense transcript expression was undetectable by Northern hybridization in U87-MG cells, which overexpress the bFGF sense mRNA. The reciprocal relationship between bFGF sense and antisense expression suggests that antisense transcripts may regulate bFGF expression in mammalian cells, and that disruption of normal sense/antisense mRNA ratios may lead to overexpression of bFGF in some tumors.

Animals↗
PubMed1992

RNA processing modulates the expression of the arcDABC operon in Pseudomonas aeruginosa.

Anaerobic growth of Pseudomonas aeruginosa on arginine depends on the arcDABC operon encoding the enzymes of the arginine deiminase pathway. The co-ordinate, anaerobic induction of these enzymes requires the FNR-like regulatory protein ANR, which activates the arc promoter lying upstream from arcD. By Northern hybridization experiments, three abundant arcA, arcAB and arcABC transcripts and three minor arcDA, arcDAB and arcDABC transcripts could be detected. The 5' ends of the arcA, arcAB and arcABC mRNAs were determined by S1 and primer extension mapping. These 5' ends appear to be generated by endonucleolytic cleavage (processing) in arcD mRNA rather than by a second promoter; this was concluded from the effects of insertion and deletion mutations in arcD. Intergenic inverted repeats between arcA and arcB as well as between arcB and arcC were shown to be involved in the formation of 3' ends of arc transcripts. Deletion of either intergenic region in the P. aeruginosa chromosome led to the loss of the arcA or arcAB transcript, respectively. Dot blot experiments revealed that arc mRNAs extracted from the wild-type strain had similar chemical half-lives in the arcA, arcB and arcC regions, ranging from 16 to 13 minutes. The half-life of arcD mRNA, by contrast, was significantly shorter, suggesting that this mRNA segment may be destabilized by the processing cuts within arcD. Deletion of the putative intergenic stem-loop structures did not result in a dramatic loss of arc mRNA stability. Thus, the intergenic hairpin structures do not contribute importantly to the overall mRNA stability; they might act primarily as partial transcription terminators and locally protect the 3' ends from exonuclease action. The expression levels of the four Arc proteins correlated approximately with the relative abundance of the corresponding mRNA segments. In conclusion, mRNA processing and, presumably, partial termination of transcription contribute to differential gene expression within the arc operon.

Amino Acid Transport Systems↗