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At least 253 records · Page 14Linked to original sources
PubMed2006

ZBP1 regulates mRNA stability during cellular stress.

An essential constituent of the integrated stress response (ISR) is a reversible translational suppression. This mRNA silencing occurs in distinct cytoplasmic foci called stress granules (SGs), which transiently associate with processing bodies (PBs), typically serving as mRNA decay centers. How mRNAs are protected from degradation in these structures remains elusive. We identify that Zipcode-binding protein 1 (ZBP1) regulates the cytoplasmic fate of specific mRNAs in nonstressed cells and is a key regulator of mRNA turnover during the ISR. ZBP1 association with target mRNAs in SGs was not essential for mRNA targeting to SGs. However, ZBP1 knockdown induced a selective destabilization of target mRNAs during the ISR, whereas forced expression increased mRNA stability. Our results indicate that although targeting of mRNAs to SGs is nonspecific, the stabilization of mRNAs during cellular stress requires specific protein-mRNA interactions. These retain mRNAs in SGs and prevent premature decay in PBs. Hence, mRNA-binding proteins are essential for translational adaptation during cellular stress by modulating mRNA turnover.

Animals↗
PubMed1989

Regulation of expression of the gene encoding the major surfactant protein (SP-A) in human fetal lung in vitro. Disparate effects of glucocorticoids on transcription and on mRNA stability.

We previously observed that dexamethasone had a biphasic effect on the levels of mRNA encoding the major surfactant protein (SP-A) in human fetal lung in vitro; at concentrations of 10(-10) and 10(-9) M, dexamethasone caused an increase in the levels of SP-A mRNA, whereas at concentrations of greater than 10(-8) M, the steroid had a pronounced inhibitory effect on SP-A mRNA levels. It was also found that dexamethasone antagonized the stimulatory effect of dibutyryl cyclic AMP (Bt2cAMP) on SP-A mRNA levels in human fetal lung in vitro. It was our objective, in the present study, to characterize further the effects of dexamethasone and Bt2cAMP on SP-A mRNA levels in human fetal lung tissue and to determine whether such effects are associated with comparable changes in the transcriptional activity of the SP-A gene. We found that the action of dexamethasone (10(-7) M) to reduce the levels of SP-A mRNA in control and Bt2cAMP-treated fetal lung explants was evident within 2 h of its addition to the culture medium; SP-A mRNA was reduced to barely detectable levels in control and in Bt2cAMP-treated tissues after 24 h of dexamethasone treatment. The action of dexamethasone to reduce SP-A mRNA levels was not prevented by co-incubation with either actinomycin D or cycloheximide. In contrast to its dose-related biphasic effects on the levels of SP-A mRNA, we found that dexamethasone caused a dose-dependent stimulation of SP-A gene transcription. Bt2cAMP also increased the transcriptional activity of the SP-A gene in the human fetal lung in vitro. In fetal lung explants incubated in the presence of dexamethasone plus Bt2cAMP, a synergistic induction of SP-A gene transcription was observed at concentrations of dexamethasone of 10(-9)-10(-7) M. Our findings are indicative that the stimulatory effects of dexamethasone (10(-10)-10(-9) M) on SP-A mRNA levels are reflective of a stimulatory effect of the steroid on SP-A gene transcription, whereas the inhibitory effects of dexamethasone (10(-7) M) on SP-A mRNA levels are the result of a dominant effect of the steroid in elevated concentrations to reduce SP-A mRNA stability.

Bucladesine↗
PubMed1999

Calcineurin enhances acetylcholinesterase mRNA stability during C2-C12 muscle cell differentiation.

Treatment of C2-C12 mouse myoblasts with the immunosuppressant drug cyclosporin A (CsA) enhances the increase in acetylcholinesterase (AChE) expression observed during skeletal muscle differentiation. The enhanced AChE expression is due primarily to increased mRNA stability because CsA treatment increases the half-life of AChE mRNA, but not the apparent transcriptional rate of the gene. Neither tacrolimus (FK506), an immunosuppressive agent with a distinct structure, nor cyclosporine H, an inactive congener of CsA, alters AChE expression. The enhanced AChE expression is associated with the muscle differentiation process, but cannot be triggered by CsA exposure before differentiation. Myoblasts and myotubes of C2-C12 cells express similar amounts of cyclophilin A and FKBP12, immunophilins known to be intracellular-binding targets for CsA and tacrolimus, respectively. However, cellular levels of calcineurin, a calcium/calmodulin-dependent phosphatase known to be the cellular target of ligand-immunophilin complexes, increase 3-fold during myogenesis. Overexpression of constitutively active calcineurin in differentiating cells reduces AChE mRNA levels and CsA antagonizes such an inhibition. Conversely, overexpression of a dominant negative calcineurin construct increases AChE mRNA levels, which are further enhanced by CsA. Thus, a CsA sensitive, calcineurin mediated pathway appears linked to differentiation-induced stabilization of AChE mRNA during myogenesis.

Acetylcholinesterase↗
PubMed2004

Increased glutathione S-transferase P1-1 expression by mRNA stabilization in hemin-induced differentiation of K562 cells.

GSTP1-1 gene expression mechanisms were investigated in hemin-induced erythroid differentiation of K562 cells. Hemoglobin production during differentiation was followed by a significant increase in GSTP1-1 mRNA (1.7-fold, P < 0.01) and protein (1.2-fold, P < 0.01) after 4 days of induction. This increase in mRNA production was not due to transcriptional up-regulation by GATA-1 previously shown to regulate GSTP1-1 during erythroid and megakaryocytic differentiation. Moreover, a drastic decrease in differentiation-specific GATA-1 mRNA expression was correlated to a reduction in GATA-1 promoter binding activity. Neither AP-1 nor NF-kappaB transcription factor binding activities could provide an explanation to the GSTP1-1 mRNA overexpression in hemin-treated cells. GSTP1-1 mRNA stability analysis using actinomycin D as an inhibitor of mRNA neosynthesis showed that mRNA half-life was doubled in hemin-induced erythroid differentiation of K562 cells. These results allow us to add stabilization of GSTP1-1 mRNA as a novel regulatory mechanism during hemin-mediated differentiation of K562 cells.

Acyltransferases↗
PubMed2000

The poly(A)-binding protein and an mRNA stability protein jointly regulate an endoribonuclease activity.

We previously identified a sequence-specific erythroid cell-enriched endoribonuclease (ErEN) activity involved in the turnover of the stable alpha-globin mRNA. We now demonstrate that ErEN activity is regulated by the poly(A) tail. The unadenylated alpha-globin 3' untranslated region (3'UTR) was an efficient substrate for ErEN cleavage, while the polyadenylated 3'UTR was inefficiently cleaved in an in vitro decay assay. The influence of the poly(A) tail was mediated through the poly(A)-binding protein (PABP) bound to the poly(A) tail, which can inhibit ErEN activity. ErEN cleavage of an adenylated alpha-globin 3'UTR was accentuated upon depletion of PABP from the cytosolic extract, while addition of recombinant PABP reestablished the inhibition of endoribonuclease cleavage. PABP inhibited ErEN activity indirectly through an interaction with the alphaCP mRNA stability protein. Sequestration of alphaCP resulted in an increase of ErEN cleavage activity, regardless of the polyadenylation state of the RNA. Using electrophoretic mobility shift assays, PABP was shown to enhance the binding efficiency of alphaCP to the alpha-globin 3'UTR, which in turn protected the ErEN target sequence. Conversely, the binding of PABP to the poly(A) tail was also augmented by alphaCP, implying that a stable higher-order structural network is involved in stabilization of the alpha-globin mRNA. Upon deadenylation, the interaction of PABP with alphaCP would be disrupted, rendering the alpha-globin 3'UTR more susceptible to endoribonuclease cleavage. The data demonstrated a specific role for PABP in protecting the body of an mRNA in addition to demonstrating PABP's well-characterized effect of stabilizing the poly(A) tail.

3' Untranslated Regions↗
PubMed2005

Glucose deprivation increases mRNA stability of vascular endothelial growth factor through activation of AMP-activated protein kinase in DU145 prostate carcinoma.

The induction of proangiogenic cytokines such as vascular endothelial growth factor (VEGF) is a critical feature of tumor angiogenesis. In the present study, we examined the mechanisms of VEGF gene expression induced by glucose deprivation in cancer cells, a role of AMP-activated protein kinase (AMPK) in the process, and the signal transduction pathway. AMPK functions as an energy sensor to provide metabolic adaptation under ATP-depleting conditions such as hypoxia and nutritional deprivation. Here, we show that glucose deprivation leads to a significant increase in the mRNA level of VEGF, GLUT1, and PFKFB3 genes in several cancer cells via a hypoxia-inducible factor-1-independent mechanism, and we demonstrate an essential role of AMPK in these gene expressions. Our data suggest that VEGF mRNA induction by glucose deprivation is due to an increase in mRNA stability, and the AMPK activity is necessary and sufficient to confer the stability to VEGF mRNA. We further show that reactive oxygen species is involved in glucose deprivation-induced AMPK activity in DU145 human prostate carcinomas, and c-Jun amino-terminal kinase acts as an upstream component in AMPK activation cascades under these conditions. LKB1, which was recently identified as a direct upstream kinase of AMPK, was not detected in DU145 cells. In conclusion, our results demonstrate a novel and major role of AMPK in the post-transcriptional regulation of VEGF, further implying its potential role in tumor angiogenesis.

AMP-Activated Protein Kinases↗
PubMed1999

Identification of a large region of secondary structure in the 3'-untranslated region of chicken elastin mRNA with implications for the regulation of mRNA stability.

Synthesis of aortic elastin peaks in the perinatal period and then is strongly down-regulated with postnatal vascular development. Our laboratory has previously shown that changes in elastin mRNA stability contribute to this developmental decrease in elastin production. Here we identify a large region of stable secondary structure in the 3'-untranslated region (3'-UTR) of chicken elastin mRNA. Reverse transcriptase polymerase chain reaction or polymerase chain reaction amplification of the 3'-UTR consistently resulted in products with an approximately 328-bp deletion from the central region of the 3'-UTR, suggesting the presence of secondary structure. The presence of this structure was confirmed by probing the 3'-UTR with RNases with selectivity for single- or double-stranded RNA. Gel migration shift assays using cytosolic extracts from 2-day old chicken aorta demonstrate specific binding of a cytosolic protein to riboprobes containing the 3'-UTR of elastin but not to riboprobes either corresponding to other areas of the message or containing the 3'-UTR but lacking the region of secondary structure. Binding of cytosolic protein was particularly prominent in aortic extracts from 2-day old chickens, a time when elastin message is stable, as compared with 8- and 15-week old chickens, when the elastin message is relatively unstable, suggesting that this region of secondary structure may play a role in developmental regulation of stability of elastin mRNA.

Animals↗
PubMed1985

Control of insulin gene expression in pancreatic beta-cells and in an insulin-producing cell line, RIN-5F cells. II. Regulation of insulin mRNA stability.

The half-life of insulin mRNA at various glucose concentrations was determined by filter hybridization techniques in isolated rat islets incubated with 3H-labeled uridine followed by a chase incubation at 3.3 or 17 mM glucose. High glucose induced a greater stabilization of insulin mRNA than of other poly(A) + RNAs or total cellular RNA. In RIN-5F insulinoma cells, an insulin-producing cell line, cholera toxin, but not glucose, induced a stabilization of insulin mRNA. After 24 h of culture of islets with actinomycin D or alpha-amanitin at several glucose concentrations, insulin mRNA content was decreased in comparison to controls only at higher glucose concentrations. The biosynthesis of islet proteins other than insulin was strongly decreased by actinomycin D at all glucose concentrations. Insulin biosynthesis was inhibited proportionately to the observed decreases in insulin mRNA content. We conclude that inhibition of insulin mRNA degradation is an important component in increasing the insulin mRNA content in response to glucose, thereby augmenting the effects of glucose stimulation on insulin gene transcription (5). This stabilization may be partly mediated by cAMP as evidenced by the similar responses to cholera toxin in the RIN-5F cells. Furthermore, the results of experiments with actinomycin D suggest that the degradation of insulin mRNA may require the continuous production of a factor(s) which could be either RNA or protein in nature.

Amanitins↗
PubMed1995

An episomal expression vector system for monitoring sequence-specific effects on mRNA stability in human cell lines.

A plasmid expression system has been developed which allows sequence-specific effects on mRNA degradation rates to be determined. This system uses stable, nonintegrating vectors that provide consistent levels of mRNA expression without the position effects common to integrating vectors. cDNAs encoding putative instability elements may be subcloned into the 5' untranslated region (5'UTR), the coding region, or the proximal 3'UTR of a beta-globin cDNA reporter. The effects of these sequences on mRNA stability may then be determined by actinomycin time course analyses of the fusion mRNAs and recombinant beta-globin mRNA in human cell lines. To demonstrate the utility of the vector system we fused an 820-bp fragment of the cDNA encoding the proximal 3'UTR of human 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase to the 3'UTR of the beta-globin reporter and introduced the vector into the human hepatocarcinoma cell line, HepG2. The fusion mRNA was degraded at a rate 2- to 2.5-fold greater than that of beta-globin alone, at a rate similar to that reported for HMG CoA reductase mRNA in normal rat liver. Similar to a number of other relatively unstable mRNAs, the rate of fusion mRNA degradation was greatly decreased by treatment with cycloheximide.

Base Sequence↗
PubMed2006

Estrogen does not regulate CD154 mRNA stability in systemic lupus erythematosus T cells.

Previous studies in our laboratory showed a dose-dependent and hormone-specific increase in CD154 expression in T cells from females with systemic lupus erythematosus (SLE). This present study investigates if the estrogen-dependent increase in CD154 expression is due to stabilization of the messenger RNA. T cells from female SLE patients and controls were cultured for 18 h in serum-free medium without and with estradiol 17-beta (10(-7) M). T cells were either unstimulated (resting) or were activated by further culture on anti-CD3 coated plates. Actinomycin D (25 microg/mL) was added to parallel cultures to inhibit new messenger RNA synthesis. CD154 messenger RNA stability was assessed by reverse-transcription polymerase chain amplification. Resting SLE (n = 10, P = 0.88) and normal (n = 7, P = 0.65) T cells showed no significant differences in message stability in response to estradiol. CD154 messenger RNA was also not significantly stabilized in activated SLE (n = 10, P = 0.15) or activated normal (n = 6, P = 0.077) T cells in response to estradiol. These findings indicate that the estrogen-dependent increase in CD154 in SLE T cells is not due to stability of the mRNA. These data are consistent with the postulate that estradiol stimulates CD154 transcription in SLE T cells.

Adult↗
PubMed2000

Cyclic nucleotide regulation of Na+/glucose cotransporter (SGLT1) mRNA stability. Interaction of a nucleocytoplasmic protein with a regulatory domain in the 3'-untranslated region critical for stabilization.

Expression of the Na(+)-coupled glucose cotransporter SGLT1 is regulated post-transcriptionally at the level of mRNA stability. We have previously demonstrated that cAMP-dependent stabilization of the SGLT1 message was correlated with the protein phosphorylation-dependent binding of cytoplasmic proteins to a uridine-rich sequence (URE) in the 3'-untranslated region (UTR). In the present study, the regulatory role of the URE was demonstrated by inserting it into the 3'-UTR of a beta-globin reporter minigene under the control of the tetracycline-regulated promoter. The resultant chimeric globin/SGLT1 mRNA expressed after transfection into LLC-PK1 cells exhibited a decreased half-life compared with the beta-globin control, indicating that the URE serves a destabilizing function. Activation of protein kinase A stabilized the chimeric message but not the beta-globin control, indicating the presence of a regulatory stabilizing sequence within the URE. A 38-kDa nucleocytoplasmic protein was identified that recognized a 12-nucleotide binding site within the URE. A mutation in this binding site that prevented protein binding assayed in vitro by UV cross-linking also prevented protein kinase A-dependent stabilization of the chimeric message assayed in vivo. These findings identify the interaction between a 38-kDa nucleocytoplasmic protein and a regulatory uridine-rich sequence in the 3'-UTR as critical for cAMP-mediated SGLT1 message stabilization.

1-Methyl-3-isobutylxanthine↗
PubMed2005

Profiling condition-specific, genome-wide regulation of mRNA stability in yeast.

The steady-state abundance of an mRNA is determined by the balance between transcription and decay. Although regulation of transcription has been well studied both experimentally and computationally, regulation of transcript stability has received little attention. We developed an algorithm, MatrixREDUCE, that discovers the position-specific affinity matrices for unknown RNA-binding factors and infers their condition-specific activities, using only genomic sequence data and steady-state mRNA expression data as input. We identified and computationally characterized the binding sites for six mRNA stability regulators in Saccharomyces cerevisiae, which include two members of the Pumilio-homology domain (Puf) family of RNA-binding proteins, Puf3p and Puf4p. We provide computational and experimental evidence that regulation of mRNA stability by these factors is modulated in response to a variety of environmental stimuli.

Gene Expression Profiling↗
PubMed1987

Herpes simplex virus virion stimulatory protein mRNA leader contains sequence elements which increase both virus-induced transcription and mRNA stability.

To investigate the role of 5' noncoding leader sequence of herpes simplex virus type 1 (HSV-1) mRNA in infected cells, the promoter for the 65,000-dalton virion stimulatory protein (VSP), a beta-gamma polypeptide, was introduced into plasmids bearing the chloramphenicol acetyltransferase (cat) gene together with various lengths of adjacent viral leader sequences. Plasmids containing longer lengths of leader sequence gave rise to significantly higher levels of CAT enzyme in transfected cells superinfected with HSV-1. RNase T2 protection assays of CAT mRNA showed that transcription was initiated from an authentic viral cap site in all VSP-CAT constructs and that CAT mRNA levels corresponded to CAT enzyme levels. Use of cis-linked simian virus 40 enhancer sequences demonstrated that the effect was virus specific. Constructs containing 12 and 48 base pairs of the VSP mRNA leader gave HSV infection-induced CAT activities intermediate between those of the leaderless construct and the VSP-(+77)-CAT construct. Actinomycin D chase experiments demonstrated that the longest leader sequences increased hybrid CAT mRNA stability at least twofold in infected cells. Cotransfection experiments with a cosmid bearing four virus-specified transcription factors (ICP4, ICP0, ICP27, and VSP-65K) showed that sequences from -3 to +77, with respect to the viral mRNA cap site, also contained signals responsive to transcriptional activation.

Acetyltransferases↗
PubMed2000

Effects of retinoic acid on N-glycosylation and mRNA stability of the liver/bone/kidney alkaline phosphatase in neuronal cells.

Alkaline phosphatase (ALP) is a glycoenzyme that is highly expressed during carcinogenesis and is induced by retinoic acid (RA) in various cells. We investigated the effects of RA on N-linked glycosylation of the tissue nonspecific liver/bone/kidney- type of ALP (L/B/K ALP), on ALP transcripts, and on total protein glycosylation in two neuronal cell lines, P19 and NG108CC15, and in primary cultures of neonatal rat brain. ALP activity was determined in cell extracts and found to be induced by RA. Tunicamycin was used at various concentrations to inhibit protein N-glycosylation. After treatment of cells with low concentrations (0.1 and 0.125 microgram/ml) of tunicamycin for 48 h, uninduced and RA-induced ALP activity declined while incubation with a protease inhibitor restored activity, indicating that the L/B/K ALP bear N-linked oligosaccharide chains important for maintaining enzymatic activity. Interestingly, ALP activity in RA-treated cultures was less inhibited by tunicamycin compared to untreated controls suggesting that RA may have an impact on ALP N-glycosylation. To investigate effects of RA on ALP glycosylation further, incorporation of [(14)C]mannose and [(35)S]methionine into ALP protein was determined in the presence or absence of RA. The ratio of mannosylation and biosynthesis demonstrate that incubation of cells with RA increased [(14)C]mannose incorporation into ALP molecules. Also, the release of free [(14)C]mannose from ALP molecules relative to the amount of protein by N-Glycanase was increased in RA-treated cultures. In addition, mannosylation of total protein was found to be induced in cells after exposure to RA. Analysis of biosynthesized ALP monomers revealed that RA increased glycosylation of the polypeptides. Furthermore, tunicamycin decreased the stability of ALP mRNA, an effect that was reduced by cotreatment with RA. Thus, the degree of N-glycosylation of the L/B/K ALP as well as mRNA and protein levels of this enzyme are affected by RA. The P19 cell line provides a useful model system to study the molecular mechanism(s) underlying the action of RA on glycosylation during neuronal differentiation further.

Alkaline Phosphatase↗
PubMed1998

Requirement for geranylgeranyl transferase I and acyl transferase in the TGF-beta-stimulated pathway leading to elastin mRNA stabilization.

The TGF-betas are multipotent in their biological activity, modulating cell growth and differentiation as well as extracellular matrix deposition and degradation. Most of these activities involve modulation of gene transcription. However, TGF-beta1 has been shown previously to substantially increase the expression of elastin by stabilization of tropoelastin mRNA through a signaling pathway which involves a phosphatidylcholine-specific phospholipase and a protein kinase C. The present results, through the use of specific inhibitors of geranylgeranyl transferase I, farnesyl transferase, and acyl transferase, demonstrate that geranylgeranylated and acylated, but not farnesyslated protein(s) is required for this TGF-beta1 effect. In addition, the general tyrosine kinase inhibitor genistein completely blocked this TGF-beta1 effect. The results suggest that the TGF-beta1 signaling pathway requires not only receptor ser/thr kinase activity, but also tyrosine kinase and small GTPase activities.

Acyltransferases↗
PubMed1993

Accelerated poly(A) loss and mRNA stabilization are independent effects of protein synthesis inhibition on alpha-tubulin mRNA in Chlamydomonas.

In Chlamydomonas, the usual rapid degradation of tubulin mRNAs induced by flagellar amputation is prevented by inhibition of protein synthesis with cycloheximide. Evidence is presented that the ability of cycloheximide to stabilize alpha-tubulin mRNA depends on the time of addition. Addition of cycloheximide to cells before induction strongly stabilizes the induced mRNAs, while addition after their synthesis stabilizes them only transiently. Moreover, cycloheximide inhibition does not stabilize the same alpha-tubulin mRNA species in uninduced cells. These results suggest that cycloheximide is not acting to stabilize the induced alpha-tubulin mRNAs simply by preventing ribosome translocation. The stabilized state of tubulin mRNA was found to correlate with its occurrence on smaller polysomes but larger EDTA-released mRNP particles than the unstable state. A second effect of cycloheximide on the metabolism of induced tubulin mRNAs is to accelerate complete poly(A) removal. This effect of cycloheximide inhibition, unlike stabilization, occurs whenever cycloheximide is added to cells, and appears unrelated to stabilization. The effect is shown to be mRNA-specific; poly(A)-shortening on the rbcS2 mRNA is not altered in the presence of cycloheximide, nor do completely deadenylated molecules accumulate. Experiments in which cells were released from cycloheximide inhibition suggest that deadenylated alpha-tubulin mRNAs may be less stable than their polyadenylated counterparts during active translation.

Animals↗
PubMed1996

mRNA stabilization in continuous flow translation system.

In contrast with standard in vitro translation systems, where 1 to 2 copies of polypeptide per mRNA molecule are produced, the continuous flow cell-free translation system is able to synthesize hundreds of polypeptide molecules per one mRNA molecule. Our investigations have shown that the poor yield obtained in the standard analytical system is due to rapid mRNA decay as opposed feedback inhibition by low molecular weight translation by products. In contrast, continuous flow system was found to stabilize mRNA for up to two-three days. RNAse activity can not be removed from wheat germ extract unless mRNA is added and compartmentalization of the translational machinery occurs.

Cell-Free System↗
PubMed2002

The control of mRNA stability in response to extracellular stimuli.

Regulated mRNA turnover is a highly important process in control of gene expression. The specific sequence elements in mRNA modulate the stability of different mRNAs, which varies considerably in response to extracellular stimuli. But the mechanistic basis for regulation of mRNA turnover remains nebulous. Recent works indicate that several signaling pathways have been implicated in regulating the decay of specific mRNA and certain ARE binding proteins mediate rapid degradation of the mRNAs. This review provides a current knowledge of diverse extracellular signals contributing to stabilization of short-lived mRNA.

Animals↗