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At least 397 records · Page 22Linked to original sources
PubMed1999

Human renin mRNA stability is increased in response to cAMP in Calu-6 cells.

The human carcinoma-derived cell line Calu-6 has previously been demonstrated to endogenously express human renin (hREN) mRNA and to markedly increase steady-state hREN mRNA levels (100-fold after 24 hours) in response to analogues of cAMP and postreceptor activators of adenylyl cyclase such as forskolin. However, both transfection analysis using hREN promoter-reporter constructs and nuclear run-on experiments suggest that transcriptional activity alone cannot account for this level of induction. We performed primer extension, reverse transcription-polymerase chain reaction, and 3' rapid amplification of cDNA ends to compare hREN mRNA between unstimulated and forskolin-stimulated cells. We demonstrate that hREN mRNA is identical under both conditions with respect to (1) utilization of the appropriate transcription start site, (2) processing of renin mRNA, and (3) utilization of the proper polyadenylation site and length of the poly-A tail. To address the mechanism of induction caused by cAMP, we used transcriptional inhibition and measured decay of hREN mRNA before and after forskolin or phorbol ester treatment. Experiments with both actinomycin D and 5, 6-dichlororibofuranosylbenzimidazole (DRB) showed that forskolin treatment markedly stabilized hREN mRNA in Calu-6 cells. A 2.3-fold increase in hREN mRNA half-life was also observed after treatment of Calu-6 cells with phorbol ester. Experiments with DRB demonstrated a similar robust stabilization of hREN mRNA after forskolin and phorbol ester treatment. These data demonstrate that the induction in hREN mRNA in response to both cAMP and phorbol ester occurs by a mechanism involving a posttranscriptional component.

Colforsin↗
PubMed2000

A role for both Ets and C/EBP transcription factors and mRNA stabilization in the MAPK-dependent increase in p21 (Cip-1/WAF1/mda6) protein levels in primary hepatocytes.

In primary hepatocytes and HepG2 hepatoma cells, prolonged activation of the p42/44 mitogen-activated protein kinase (MAPK) pathway is associated with a reduction in DNA synthesis, mediated by increased expression of the cyclin-dependent kinase inhibitor protein p21 (Cip-1/WAF1/mda6) (p21). This study was performed to evaluate the contribution of transcriptional and post-transcriptional regulation in this response. Prolonged activation of the MAPK pathway in wild-type or p21 null hepatocytes caused a large decrease and increase, respectively, in DNA synthesis. Prolonged activation of the MAPK pathway in either wild-type or p21 antisense HepG2 cells also caused large decreases and increases, respectively, in DNA synthesis. MAPK signaling increased the phosphorylation of the transcription factors Ets2, C/EBPalpha, and C/EBPbeta, and rapidly increased transcription from the p21 promoter via multiple Ets- and C/EBP-elements within the enhancer region. Eight hours after MAPK activation, loss of C/EBPbeta or Ets2 function significantly reduced MAPK-stimulated transcription from the p21 promoter and abolished increased p21 protein expression. At this time, MAPK signaling increased both p21 mRNA and p21 protein stabilities that were also demonstrated to be essential for a profound increase in p21 protein levels. Thirty-six hours after MAPK activation, transcription from the p21 promoter was still significantly reduced in cells without either C/EBPbeta or Ets2 function; however, these cells were now capable of exhibiting a partial increase in p21 protein expression. In contrast, loss of C/EBPalpha function modestly reduced MAPK-stimulated transcription from the p21 promoter but strongly inhibited the ability of prolonged MAPK activation to increase protein levels of p21. This data suggested that prolonged enhancement of p21 protein levels may be under posttranscriptional control. In agreement with this hypothesis, prolonged MAPK signaling further increased p21 mRNA stability at 36 h, compared with the 8-h time point. Our data argue that MAPK signaling increased p21 promoter activity via multiple transcription factors, which alone were insufficient for a robust prolonged increase in p21 protein levels in primary hepatocytes, and that to increase p21 protein levels also required enhanced stabilization of p21 mRNA and p21 protein. Collectively, these data suggest that loss of transcription factor and mRNA/protein stabilization functions correlates with an inability of MAPK signaling to cause growth arrest versus proliferation in primary hepatocytes.

Animals↗
PubMed1996

A dual role for the Bacillus subtilis glpD leader and the GlpP protein in the regulated expression of glpD: antitermination and control of mRNA stability.

The Bacillus subtilis glpD gene encodes glycerol-3-phosphate dehydrogenase. This gene is preceded by a leader region containing an inverted repeat which acts as a transcription terminator. Expression of glpD is controlled by antitermination of transcription at the inverted repeat. Antitermination is effected by the glpP gene product in conjunction with glycerol-3-phosphate and, consequently, GlpP mutants fail to grow on glycerol as a sole carbon and energy source. We have isolated a number of glycerol-positive revertants of GlpP mutants. Most of these revertants have mutations in the inverted repeat of the glpD leader and produce glycerol-3-phosphate dehydrogenase constitutively. Unlike wild-type bacteria, they are not sensitive to glucose repression of glpD. A few of the revertants are temperature sensitive, i.e. they grow on glycerol at 32 degrees C but not at 45 degrees C and produce glycerol-3-phosphate dehydrogenase only at 32 degrees C. Northern blot analyses demonstrated that the temperature-sensitive expression of glpD is due to destabilization of glpD mRNA. Furthermore, introduction of the wild-type glpP gene into the revertants stabilized the glpD mRNA. This is probably a result of a direct interaction between the GlpP protein and the leader of glpD mRNA. Besides its function in antitermination of transcription of glpD, it is suggested that GlpP is also involved in controlling glpD mRNA stability. Introduction of the glpP gene into the revertants also restored glucose repression, indicating that this repression is mediated by the GlpP protein.

Bacillus subtilis↗
PubMed2000

Regulation of tumour necrosis factor alpha mRNA stability by the mitogen-activated protein kinase p38 signalling cascade.

The translation of tumour necrosis factor alpha (TNFalpha) mRNA is regulated by the stress-activated protein kinase p38, which also controls the stability of several pro-inflammatory mRNAs. The regulation of TNFalpha gene expression in a mouse macrophage cell line RAW264.7 was re-examined using an inhibitor of stress-activated protein kinases. Stimulation of these cells with bacterial lipopolysaccharide resulted in stabilisation of TNFalpha mRNA, which was reversed by specific inhibition of p38. An adenosine/uridine-rich element from the TNFalpha 3' untranslated region conferred p38-sensitive decay in a tetracycline-regulated mRNA stability assay. Therefore the p38 pathway also controls TNFalpha mRNA turnover.

3' Untranslated Regions↗
PubMed1998

Hsp70 accumulation in chondrocytic cells exposed to high continuous hydrostatic pressure coincides with mRNA stabilization rather than transcriptional activation.

In response to various stress stimuli, heat shock genes are induced to express heat shock proteins (Hsps). Previous studies have revealed that expression of heat shock genes is regulated both at transcriptional and posttranscriptional level, and the rapid transcriptional induction of heat shock genes involves activation of the specific transcription factor, heat shock factor 1 (HSF1). Furthermore, the transcriptional induction can vary in intensity and kinetics in a signal- and cell-type-dependent manner. In this study, we demonstrate that mechanical loading in the form of hydrostatic pressure increases heat shock gene expression in human chondrocyte-like cells. The response to continuous high hydrostatic pressure was characterized by elevated mRNA and protein levels of Hsp70, without activation of HSF1 and transcriptional induction of hsp70 gene. The increased expression of Hsp70 was mediated through stabilization of hsp70 mRNA molecules. Interestingly, in contrast to static pressurization, cyclic hydrostatic loading did not result in the induction of heat shock genes. Our findings show that hsp70 gene expression is regulated posttranscriptionally without transcriptional induction in chondrocyte-like cells upon exposure to high continuous hydrostatic pressure. We suggest that the posttranscriptional regulation in the form of hsp70 mRNA stabilization provides an additional mode of heat shock gene regulation that is likely to be of significant importance in certain forms of stress.

Cartilage↗
PubMed2002

Tetracycline induces stabilization of mRNA in Bacillus subtilis.

The tet(L) gene of Bacillus subtilis confers low-level tetracycline (Tc) resistance. Previous work examining the >20-fold-inducible expression of tet(L) by Tc demonstrated a 12-fold translational induction. Here we show that the other component of tet(L) induction is at the level of mRNA stabilization. Addition of a subinhibitory concentration of Tc results in a two- to threefold increase in tet(L) mRNA stability. Using a plasmid-borne derivative of tet(L) with a large in-frame deletion of the coding sequence, the mechanism of Tc-induced stability was explored by measuring the decay of tet(L) mRNAs carrying specific mutations in the leader region. The results of these experiments, as well as experiments with a B. subtilis strain that is resistant to Tc due to a mutation in the ribosomal S10 protein, suggest different mechanisms for the effects of Tc on translation and on mRNA stability. The key role of the 5' end in determining mRNA stability was confirmed in these experiments. Surprisingly, the stability of several other B. subtilis mRNAs was also induced by Tc, which indicates that addition of Tc may result in a general stabilization of mRNA.

5' Untranslated Regions↗
PubMed1991

Posttranscriptional regulation of coumarin 7-hydroxylase induction by xenobiotics in mouse liver: mRNA stabilization by pyrazole.

The induction mechanism by pyrazole or phenobarbital of coumarin 7-hydroxylase (cytochrome P450coh) was investigated in DBA/2J male mice. The P450coh mRNA in the pyrazole-induced mice was increased gradually to a 20-fold higher level within 48 h, yet transcription of the P450coh gene was not affected. The half-life of P450coh mRNA, on the other hand, was at least 4-fold longer in the pyrazole-induced DBA/2J (approximately 6.0 h) than in control DBA/2J (approximately 1.5 h) male mice. The stabilization of P450coh mRNA, therefore, is the primary mechanism for the induction by pyrazole of coumarin 7-hydroxylase. Phenobarbital, on the other hand, regulates the induction either translationally or posttranslationally. This drug affected neither the P450coh mRNA nor the P450coh gene's transcription levels in the DBA/2J male mice, although Western blots showed approximately a 3-fold increase of the P450coh protein in the liver microsomes of the drug-treated mice. The results indicate, therefore, that both phenobarbital and pyrazole regulate the P450coh induction posttranscriptionally; the former inducer enhances the translational efficiency of P450coh mRNA or alters the degradation rate of P450coh aproprotein, while the latter stabilizes P450coh mRNA.

Animals↗
PubMed1993

Continuous presence of phorbol ester is required for its IL-1 beta mRNA stabilizing effect.

The protein kinase C (PKC) activating phorbol esters are known to prevent the decay of mRNA of several cytokines and proto-oncogenes. To examine whether the phorbol ester signal is continuously required for this stabilizing effect, THP-1 monocytic cells were stimulated either with phorbol 12,13-dibutyrate (PDBu), which can be removed from the cells by washings, or with the more hydrophobic phorbol 12-myristate 13-acetate (PMA). Both of these stimuli induced high levels of interleukin-1 beta (IL-1 beta) mRNA. When the cells were washed at the peak of the IL-1 beta mRNA expression, this mRNA decayed rapidly in the PDBu stimulated cells while in PMA stimulated cells the mRNA levels were not affected. Moreover, this mRNA degradation induced by the removal of PDBu could be inhibited by readdition of the phorbol ester. This restabilization could be prevented by pharmacologic inhibitors of PKC, but not by inhibiting protein or RNA synthesis. Thus these data suggest that the phorbol ester must be continuously present to exert its mRNA stabilizing effect and that its effect is PKC-mediated but does not require active protein or RNA synthesis.

Cells, Cultured↗
PubMed2004

Stimulus-specific induction of a novel nuclear factor-kappaB regulator, IkappaB-zeta, via Toll/Interleukin-1 receptor is mediated by mRNA stabilization.

We have recently identified an inducible nuclear factor-kappaB (NF-kappaB) regulator, IkappaB-zeta, which is induced by microbial ligands for Toll-like receptors such as lipopolysaccharide and the proinflammatory cytokine interleukin (IL)-1beta but not by tumor necrosis factor (TNF)-alpha. In the present study, we examined mechanisms for stimulus-specific induction of IkappaB-zeta. The analysis of the IkappaB-zeta promoter revealed an essential role for an NF-kappaB binding sequence in transcriptional activation. The activation, however, did not account for the Toll-like receptor/IL-1 receptor-specific induction of IkappaB-zeta, because the promoter analysis and nuclear run-on analysis indicated that its transcription was similarly induced by TNF-alpha. To examine post-transcriptional regulation, we analyzed the decay of IkappaB-zeta mRNA, and we found that it was specifically stabilized by lipopolysaccharide or IL-1beta but not by TNF-alpha. Furthermore, we found that costimulation with TNF-alpha and another proinflammatory cytokine, IL-17, elicited the IkappaB-zeta induction. Stimulation with IL-17 alone did not induce IkappaB-zeta but stabilized its mRNA. Therefore, IkappaB-zeta induction requires both NF-kappaB activation and stimulus-specific stabilization of its mRNA. Because IkappaB-zeta is essential for expression of a subset of NF-kappaB target genes, the stimulus-specific induction of IkappaB-zeta may be of great significance in regulation of inflammatory reactions.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Regulation of ompA mRNA stability: the role of a small regulatory RNA in growth phase-dependent control.

The Escherichia coli ompA mRNA, encoding a highly abundant outer membrane protein, has served as a model for regulated mRNA decay in bacteria. The half-life of this transcript correlates inversely with the bacterial growth rate and is growth stage-dependent. The stability of the messenger is determined by the 5'-untranslated region which possesses cleavage sites for RNase E. Hfq binds to this region, is essential for controlling the stability and has been suggested to directly regulate ompA mRNA decay. Here we report that the 78 nucleotide SraD RNA, which is highly conserved among Enterobacteriaceae, acts in destabilizing the ompA transcript when rapidly grown cells enter the stationary phase of growth. During this growth-stage the expression of SraD RNA becomes strongly increased. The SraD-mediated decay of ompA mRNA depends on Hfq and in vitro studies revealed that Hfq facilitates binding of the regulatory RNA to the translational initiation region of the messenger. Deletion of sraD, however, does not significantly affect the stability of the ompA mRNA in slowly growing cells. Our results indicate that distinct regulatory circuits are responsible for growth phase- and growth rate-dependent control of the ompA mRNA stability.

5' Untranslated Regions↗
PubMed2000

An in vitro assay to study regulated mRNA stability.

The examination of posttranscriptional regulation of mRNA in mammalian cells is critical to discovering the role that mRNA plays in the initiation and maintenance of cellular processes. The complexity of the system defies a holistic approach and, therefore, we have devised an in vitro mRNA turnover assay that enables us to elucidate the factors involved in mRNA deadenylation and degradation. Our system, using an S100 HeLa extract and in vitro transcribed RNAs, accurately mimics the end products of mRNA turnover, which have been previously described using in vivo studies and, in addition, allows for the detailed study of factors that may play a role in regulated deadenylation and degradation. Another important aspect of our system is the ease with which it can be manipulated. We can provide any synthetic RNA molecule to the assay to test for specific sequence activity. Furthermore, the results are clear and accurately interpretable. We have demonstrated that our in vitro system accurately deadenylates and decays a capped and polyadenylated RNA molecule in a processive manner without nonspecific nuclease activity. Finally, we have demonstrated regulated instability in vitro using the AU-rich elements (AREs) from tumor necrosis factor-alpha (TNF-alpha) and granulocyte macrophage colony stimulating factor (GM-CSF) embedded within the RNA molecule. The presence of the AREs increased the deadenylation and the decay rates seen in vivo. We feel that this system can be expanded and adapted to examine a variety of mRNA regulatory events in mammalian cells.

HeLa Cells↗
PubMed1996

Selective control of cytosolic glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase mRNA stability by selenium supply.

Selenium depletion of H4 hepatoma cells reduced cytosolic glutathione peroxidase (cGSH-Px) mRNA abundance but had no effect on phospholipid hydroperoxide glutathione peroxidase (PHGSH-Px) mRNA abundance. Actinomycin D chase experiments showed that selenium depletion had no effect on the stability of PHGSH-Px mRNA but decreased the stability of cGSH-Px mRNA. In Se-replete cells puromycin decreased the stability of both cGSH-Px and PHGSH-Px mRNAs. The results suggest that when selenium supply is limiting PHGSH-Px mRNA translation is maintained more than that of cGSH-Px mRNA, and thus more cGSH-Px mRNA is released from polysomes and degraded.

Animals↗
PubMed2001

A nucleus-encoded suppressor defines a new factor which can promote petD mRNA stability in the chloroplast of Chlamydomonas reinhardtii.

Mutations in the Chlamydomonas reinhardtii nuclear gene MCD1 specifically destabilize the chloroplast petD mRNA, which encodes subunit IV of the cytochrome b6/f complex. The MCD1 gene product is thought to interact with the mRNA 5' end to protect it from degradation by a 5' --> 3' exoribonuclease and may also have a role in translation initiation. Here we report the isolation and characterization of a semidominant, allele-specific, nucleus-encoded suppressor of the mcd1-2 mutation. The suppressor mutation, which defines a new locus MCD2, allows accumulation of 10% of the wild-type level of petD mRNA and as much as 50% of the wild-type subunit IV level. Taken together, these results suggest the suppressor mutation restores photosynthetic growth by stabilizing petD mRNA. In addition, it may promote increased translational efficiency, an inference supported by direct measurements of the subunit IV synthesis rate. Thus, both MCD1 and MCD2 may participate in both chloroplast RNA stability and translation initiation.

Animals↗
PubMed1994

Erythroid cell-specific determinants of alpha-globin mRNA stability.

Although globin mRNAs are considered prototypes of highly stable messages, the mechanisms responsible for their longevity remain largely undefined. As an initial step in identifying potential cis-acting elements or structures which contribute to their stability, we analyzed the defect in expression of a naturally occurring alpha 2-globin mutant, alpha Constant Spring (CS). The CS mutation is a single-base change in the translation termination codon (UAA-->CAA) that allows the ribosome to read through into the 3' nontranslated region (NTR). The presence of CS mRNA in transcriptionally active erythroid precursors and its absence (relative to normal alpha-globin mRNA) in the more differentiated transcriptionally silent erythrocytes suggest that this mutation disrupts some feature of the alpha-globin mRNA required for its stability. Using a transient transfection system, we demonstrate that in murine erythroleukemia cells the CS mRNA is unstable compared with the normal alpha 2-globin mRNA. The analyses of several other naturally occurring and site-directed mutant alpha-globin genes in murine erythroleukemia cells indicate that entry of a translating ribosome into the 3' NTR targets the message for accelerated degradation in erythroid cells. In contrast, both the CS and alpha 2-globin mRNAs are stable in several nonerythroid cell lines. These results suggest that translational readthrough disrupts a determinant associated with the alpha 2-globin 3' NTR which is required for mRNA stability in erythroid cells.

Animals↗
PubMed1990

Control of mRNA stability in chloroplasts by 3' inverted repeats: effects of stem and loop mutations on degradation of psbA mRNA in vitro.

To investigate the role of mRNA 3' inverted repeats (IRs) in stabilizing plant chloroplast mRNAs, we have measured the processing and stability of wild-type and mutant RNAs corresponding to the 3' end of the spinach chloroplast psbA mRNA. wild-type and mutant 3' IR-RNA precursors were processed at similar rates in a homologous in vitro system, but RNAs with either a mutant loop sequence CUUCGG or a specific base substitution in the IR exhibited an enhanced accumulation of mature product. Incubation of mature products in the in vitro system demonstrated that this was due to an increased stability of the product. These mutant RNAs displayed the same order of stabilities when their decay was measured following electroporation into intact chloroplasts. We found that the in vitro system contains an endonuclease activity that cleaves the wild-type 3' IR-RNA within the loop and also in single-stranded regions, suggesting a possible role for the loop sequence in determining RNA longevity in vitro. Interestingly, the altered loop sequence CUUCGG, which enhances RNA stability in bacteria (1), prolonged the half-life of psbA 3' IR-RNA in vitro and also resulted in an altered endonuclease cleavage pattern. Such nucleases could potentially play an important role in plastid mRNA decay in vivo.

Base Composition↗
PubMed2003

Neuronal HuD gene encoding a mRNA stability regulator is transcriptionally repressed by thyroid hormone.

Many genes governed by thyroid hormone (T3) lack binding sites for its receptor (TR) and are thought to be post-transcriptionally regulated by T3. Here we demonstrate that the HuD gene, which encodes a neurone-specific protein that binds to mRNA and modulates its stability, is regulated by T3. HuD RNA and protein expression were strongly up-regulated in specific areas of the hypothyroid rat brain, and reduced by T3 in rat PC12 and mouse N2a cells containing appropriate TR levels. Furthermore, T3 inhibited the transcription of HuD in run-on assays. Finally, HuD protein bound with high affinity to two sequences in acetylcholinesterase mRNA, and ectopic HuD expression increased its abundance in N2a cells. This is the first report of a gene encoding an mRNA stability regulator that is under T3 control. The results suggest that HuD may mediate some T3 effects by altering the half-life of mRNAs for acetylcholinesterase and other genes.

Acetylcholinesterase↗
PubMed1994

Developmental and hormonal regulation of surfactant protein C (SP-C) gene expression in fetal lung. Role of transcription and mRNA stability.

Pulmonary surfactant protein C (SP-C) gene expression is developmentally and hormonally regulated in fetal lung. In the present study, we investigated the role of transcriptional and posttranscriptional mechanisms in the developmental, cAMP, and dexamethasone induction of SP-C mRNA. We found that developmental induction of SP-C mRNA was not coincident with induction of SP-C gene transcription. SP-C mRNA levels reached approximately 90% of levels in adult lung on day 24 of gestation, whereas SP-C gene transcription was only approximately 4% of level in adult lung and did not increase until day 28 of gestation (term in rabbit = 31 days). Treatment of fetal lung tissues in vitro with dibutyryl cyclic AMP (Bt2cAMP) and dexamethasone increased SP-C mRNA accumulation by different mechanisms. Increase in SP-C mRNA accumulation by Bt2cAMP was the result of increased SP-C gene transcription, whereas increased SP-C mRNA accumulation by dexamethasone was due to stabilization of RNA. In control tissues the SP-C mRNA half-life (t1/2) was 11.2 h, and after dexamethasone treatment it increased to 30 h. These data show that both transcriptional and mRNA stabilization mechanisms regulate induction of SP-C gene expression during fetal lung development and by cAMP and dexamethasone in fetal lung in vitro.

Animals↗
PubMed2005

Reuptake of extracellular amelogenin by dental epithelial cells results in increased levels of amelogenin mRNA through enhanced mRNA stabilization.

Amelogenin is an extracellular matrix protein secreted by ameloblasts and is a major component of enamel matrix. Recently, in addition to their role in enamel formation, the biological activity of enamel proteins in the process of cell differentiation has recently become widely appreciated. In this study, we examined the biological activity of amelogenin on ameloblast differentiation. Recombinant mouse amelogenin (rm-amelogenin) enhanced the expression of endogenous amelogenin mRNA in a cultured dental epithelial cell line (HAT-7), despite a lack of increased amelogenin promoter activity. To solve this discrepancy, we analyzed the effects of rm-amelogenin on the stability of amelogenin mRNA. The half-life of amelogenin mRNA is extremely short, but in the presence of rm-amelogenin its half-life was extended three times longer than the control. Furthermore, we showed the entry of exogenous fluorescein isothiocyanate-conjugated rm-amelogenin into the cytoplasm of HAT-7 cells. It follows from our results that exogenous amelogenin increases amelogenin mRNA levels through stabilization of mRNA in the cytoplasm of HAT-7 cells. Here we speculated that during differentiation, dental epithelial cells utilize a unique mechanism for increasing the production of amelogenin, the reuptake of secreted amelogenin.

3' Untranslated Regions↗