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At least 487 records · Page 27Linked to original sources
PubMed1987

Effect of premature termination of translation on mRNA stability depends on the site of ribosome release.

Translational stop codons were introduced at various locations in the protein-coding regions of the monocistronic bla and ompA gene transcripts of Escherichia coli, and the decay characteristics of the upstream and downstream mRNA segments were analyzed. Premature termination of translation at codon position 26 reduced the stability of both the translated and ribosome-free segments of bla mRNA, whereas release of ribosomes just 30 codons further downstream resulted in normal stability for both segments. Normal stability of an untranslated bla gene mRNA segment required its linkage to a ribosome-bound segment of bla gene mRNA. These findings indicate that depriving an mRNA segment of ribosomes does not necessarily render it more susceptible to degradation. However, premature termination of translation at a location that allows ribosomes to traverse only a short segment of bla mRNA can lead to destabilization of the entire transcript.

Bacterial Outer Membrane Proteins↗
PubMed1999

Effect of insulin on farnesyltransferase gene transcription and mRNA stability.

Recently, we have shown that hyperinsulinemia increases the activity of farnesyltransferase (FTase) in vitro (1) and in hyperinsulinemic animals (2), stimulates the phosphorylation of the FTase alpha-subunit (3), increases the amounts of cellular farnesylated p21Ras (4), and potentiates the nuclear effects of other peptide growth factors, such as EGF, IGF-1 and PDGF (5). To further investigate the mechanism by which insulin stimulates FTase activity we tested the effect of insulin on the rate of FTase transcription, the rate of FTase mRNA degradation, and the amounts of FTase protein. Insulin increased the amounts of FTase alpha- and beta-subunit mRNA in 3T3-L1 fibroblasts 2.5-fold to 4-fold after 6 h and 24 h incubation, respectively, but did not increase the rate of FTase transcription over a 24 h period. Insulin did, however, increase the stability of both alpha- and beta-subunit mRNA. The half-life for both FTase alpha- and beta-subunit mRNA was approximately 3 h and 6h in the absence and in the presence of insulin, respectively. Although insulin stabilized the alpha- and beta-subunit mRNA of FTase, there was no increase in amounts of protein of either subunit. These data suggest that although insulin increases the stability of the FTase mRNA, it stimulates FTase enzymatic activity only at the post-translational level.

3T3 Cells↗
PubMed1993

Mammalian ribonucleotide reductase R1 mRNA stability under normal and phorbol ester stimulating conditions: involvement of a cis-trans interaction at the 3' untranslated region.

Ribonucleotide reductase R1 gene expression is elevated in BALB/c 3T3 fibroblasts treated with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). We show that TPA treatment increased the half-life of R1 mRNA by 5-fold, indicating that TPA regulates R1 gene expression by a post-transcriptional mechanism. We investigated the possibility that the 3' untranslated region (3'UTR) of R1 mRNA contains regulatory information for TPA-mediated message stability. Our studies demonstrated that a 49 nucleotide (nt) TPA-responsive region existed within the R1 mRNA 3'UTR. Deletion of the 49 nt region led to the abolishment of TPA-induced stability of R1 and hybrid CAT mRNAs. Further deletions of the 3'UTR did not significantly affect mRNA turnover rates. In addition, we detected by cross-linking a novel 52-57 kDa R1 mRNA-binding protein (R1BP) that bound selectively to the 49 nt region of the R1 mRNA 3'UTR and did not bind to the 5'UTR, the coding region or other mRNA 3'UTRs. The R1BP-RNA binding activity observed in unstimulated cells was rapidly and markedly down-regulated after TPA treatment, suggesting a role for R1BP in mRNA metabolism, and in the mechanism of action of TPA-induced R1 message stabilization. These results support a novel model of R1 gene regulation in which a cis-element(s) within the 49 nt region of the R1 mRNA 3'UTR interacts with R1BP in a mechanism that regulates R1 message stability. We propose that this model accounts for the TPA-mediated stability alteration of R1 message, through down-regulation of R1BP-RNA binding activity linked to a reduction in the rate of R1 mRNA degradation.

3T3 Cells↗
PubMed1993

Glucose deprivation causes posttranscriptional enhancement of brain capillary endothelial glucose transporter gene expression via GLUT1 mRNA stabilization.

The absence of neuroglucopenia symptoms in chronic hypoglycemia may be due to up-regulation of the blood-brain barrier glucose transporter type 1 (GLUT1). Therefore, we investigated the effect of glucose deprivation on the abundance of the GLUT1 transcript in bovine brain capillary endothelial cells in tissue culture (ECL). Northern blot analysis performed under high stringency conditions with 4-5 micrograms of ECL poly(A)+ mRNA showed that glucose deprivation (5 mg% glucose) caused a 2.4 +/- 0.2-fold increase in the GLUT1/actin mRNA ratio versus control incubations (100 mg% glucose). This rise was dose and time dependent, and the maximum effect was observed 20-24 h after the hexose deprivation. Nuclear transcription run-on assay showed no changes in either the GLUT1 or actin gene transcription rate 24 h after glucose deprivation. To determine whether the increase in the abundance of the GLUT1 mRNA induced by glucose deprivation was due to increased stability of this transcript, the GLUT1 mRNA half-life was measured in ECL cells incubated with actinomycin D. The levels of the GLUT1 transcript continued to be augmented in glucose-deprived cells compared with controls 2 and 4 h after the transcription inhibitor was added to the media. Glucose deprivation induced a 78% increase in the t1/2 of the GLUT1 mRNA (from 3.6 to 6.4 h). Incubation of ECL cells with the protein synthesis inhibitor, cycloheximide, for 4 h partially reversed the effect of glucose deprivation on the abundance of the GLUT1 transcript. On the other hand, incubation with cycloheximide for 24 h completely blocked the effect of glucose deprivation on the GLUT1 transcript. Desensitization of cellular protein kinase C was performed by incubation of ECL cells with 1 microM phorbol ester for 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1995

Identification of the cis-elements mediating the autogenous control of ribosomal protein L2 mRNA stability in yeast.

The ribosomal protein L2 (rpL2) of Saccharomyces cerevisiae regulates the accumulation of its own mRNA by a feedback mechanism. An RNA sequence is responsible for this control, initially characterized as a 360 nucleotide-long region, localized at the 5' end of the transcript. This region, fused to an unrelated coding sequence, is able to down-regulate the accumulation of the chimeric transcript when increased levels of rpL2 are induced in the cell. The target regulatory region also responds to regulation when inserted inside an intron, demonstrating that the control process can take place inside the nucleus. Deletion analysis from the 5' and 3' borders have restricted the responsive region to approximately 200 nt. The insertion of a poly-G cassette downstream of the regulatory region allowed the identification of truncated 3' cut-off poly(A)+ RNA molecules. The parallel identification of cut-off molecules containing the 5' portion of the transcript allowed us to deduce that the truncated products originate by endonucleolytic cleavage. Altogether, these results are consistent with a mechanism by which the presence of excess amounts of rpL2 in the cell triggers its own mRNA to a degradative pathway; this involves an initial endonucleolytic cleavage that is followed by exonucleolytic trimming. Such a regulatory mechanism shows interesting analogies with the translational regulation of r-proteins in Escherichia coli.

Base Sequence↗
PubMed2000

mRNA stability in eukaryotes.

During the past two years, the role of the proteins HuR and hnRNP D in regulated mRNA degradation in humans has become clearer, and a putative mRNA deadenylase, DAN or PARN, has been identified. In yeast, the relationship between translation and mRNA turnover is clearer, but the mRNA decapping process has turned out to be unexpectedly complex.

Animals↗
PubMed1990

Phage fl mRNA processing in Escherichia coli: search for the upstream products of endonuclease cleavage, requirement for the product of the altered mRNA stability (ams) locus.

In Escherichia coli infected with the filamentous phage f1, a number of the polycistronic phage mRNA species are generated through post-transcriptional processing by host nuclease activity. In this paper we review experimental evidence assessing whether known RNases are involved in mediating these processing events, and we use S1 nuclease mapping methods to visualize putative upstream products of endonuclease cleavage. By examining f1 processing in a phage-infected host bearing a temperature-sensitive allele of the altered message stability locus (ams), we show that production of the major processed species requires a component of the host cell which functions in the messenger RNA decay process.

Coliphages↗
PubMed2001

HuR and mRNA stability.

An important mechanism of posttranscriptional gene regulation in mammalian cells is the rapid degradation of messenger RNAs (mRNAs) signaled by AU-rich elements (AREs) in their 3' untranslated regions. HuR, a ubiquitously expressed member of the Hu family of RNA-binding proteins related to Drosophila ELAV, selectively binds AREs and stabilizes ARE-containing mRNAs when overexpressed in cultured cells. This review discusses mRNA decay as a general form of gene regulation, decay signaled by AREs, and the role of HuR and its Hu-family relatives in antagonizing this mRNA degradation pathway. The influence of newly identified protein ligands to HuR on HuR function in both normal and stressed cells may explain how ARE-mediated mRNA decay is regulated in response to environmental change.

Amino Acid Sequence↗
PubMed1987

The stem-loop structure at the 3' end of histone mRNA is necessary and sufficient for regulation of histone mRNA stability.

Chimeric genes were made by fusing mouse histone genes with a human alpha-globin gene. The genes were introduced into mouse L cells and the stability of the chimeric mRNAs was measured when DNA synthesis was inhibited. An mRNA containing all the globin coding sequences and the last 30 nucleotides of the histone mRNA was degraded at the same rate as histone mRNA.

Animals↗
PubMed2004

Lysophosphatidic acid inhibits TGF-beta-mediated stimulation of type I collagen mRNA stability via an ERK-dependent pathway in dermal fibroblasts.

Lysophosphatidic acid (LPA) is a serum-derived pleiotropic mediator with a potential role in wound repair. Since extracellular matrix (ECM) deposition is a critical part of wound healing, this study was designed to examine whether LPA is involved in ECM regulation. Using human dermal fibroblasts, we demonstrate that LPA counteracts transforming growth factor-beta (TGF-beta) stimulation of type I collagen mRNA and protein. This factor elicits its inhibitory effects at the posttranscriptional level via destabilization of type I collagen mRNA. Furthermore, using the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059, we show that the extracellular signal-regulated kinase (ERK) pathway is a negative regulator of the TGF-beta-induced stabilization of type I collagen mRNA, and that the activation of the ERK pathway by LPA mediates their inhibitory effects on collagen production. In conclusion, this study describes a novel function for LPA as an antagonist of TGF-beta induced ECM deposition. These findings may be relevant to physiologic wound repair and may be useful in designing therapeutic agents to prevent excessive scarring.

Blotting, Northern↗
PubMed2006

Interferon-gamma-inducible protein (IP)-10 mRNA stabilized by RNA-binding proteins in monocytes treated with S100b.

Chemokines mediate the recruitment and activation of blood monocyte/macrophages and lymphocytes to sites of inflammation. Expression of the chemokine IP-10 (interferon-gamma-inducible protein) has been documented in several inflammatory and autoimmune disorders including type 1 diabetes. However, the mechanism of its expression in monocytes or its functional role in diabetes is not known. Advanced glycation end products acting via their receptor, RAGE, play major roles in diabetic complications. In this study, we observed for the first time that S100b, an inflammatory protein as well as a specific RAGE ligand, significantly increased IP-10 mRNA and protein levels in THP-1 monocytes as well as peripheral blood monocytes. Promoter luciferase assays showed that IP-10 mRNA accumulation by S100b was not via increased transcription. On the other hand, S100b significantly increased IP-10 mRNA half-life and stability. This appeared to be mediated by S100b-induced binding of specific RNA-binding protein(s) to a 3'-untranslated region-responsive region of the IP-10 mRNA. Our results demonstrate for the first time that diabetic stimuli such as RAGE ligands can induce inflammatory gene expression in monocytes via increased message stability.

3' Untranslated Regions↗
PubMed1994

Reverse interpretation: a hypothetical selection mechanism for adaptive mutagenesis based on autoregulated mRNA stability.

The problem of adaptive mutagenesis is examined under the assumption that selection occurs through the interaction of cognate mRNA and protein molecules, a phenomenon that has been considered to lead to differential mRNA decay (Kyrpides & Ouzounis, 1993, J. theor. Biol. 163, 373-392). Based on the stereochemical hypothesis, we speculate that the origin of this process relies on an extended form of molecular interactions. This mechanism, termed reverse interpretation, predicts the selective amplification of successful molecular forms with the capability of their immortalization through reverse transcription, in a manner that resembles, but is not equivalent to, reverse translation. Finally, we argue that the proposed mechanism conforms with the Darwinian view of evolutionary change, if variability is considered to be a property of molecular and not cellular populations.

Animals↗
PubMed1997

Nerve growth factor mRNA stability is controlled by a cis-acting instability determinant in the 3'-untranslated region.

Nerve growth factor (NGF) mRNA is rapidly degraded in many non-neuronal cell types with a half-life of between 30 and 60 min. Similar to other short-lived mRNAs the 3'-untranslated region (3'-UTR) of the NGF mRNA contains a short AU nucleotide-rich sequence. To implicate this region as a cis-acting determinant of NGF mRNA instability, expression vectors containing NGF cDNA with and without the 3'-UTR, and vectors containing only the 3'-UTR were constructed and used in cell transfection experiments. Transfection of HEK293 or NIH3T3 cells with these expression vectors followed by measurement of NGF mRNA half-life indicated that NGF mRNA without the AU-rich 3'-UTR was approximately 3-fold more stable than NGF mRNA containing the 3'-UTR. Similar results were seen in a polysome-based cell-free RNA decay assay using NGF mRNA with and without the 3'-UTR prepared from transfected cells. Addition of a short RNA containing the AU-rich 3'-UTR to the cell-free RNA decay system prolonged the half-life of the full-length NGF mRNA, suggesting competition between these two RNA species for polysome-associated factors which degrade the NGF mRNA. Moreover, transfection of HEK293 or astroglial cells with vectors designed to express only the AU-rich region of the 3'-UTR resulted in enhanced expression of NGF mRNA. The results indicate that the 3'-UTR of the NGF mRNA contains a cis-acting instability determinant which, perhaps by interacting with trans-acting RNA-binding proteins, controls the rate of NGF mRNA turnover.

Animals↗
PubMed2005

[A preliminary study on estimation of postmortem interval according to beta-actin mRNA stability in rat].

OBJECTIVE: In order to find a new parameter to estimate the postmortem interval, beta-actin mRNA in lung and thoracic muscle of rats was detected at different time point postmortem. METHODS: Rats were killed by neck dislocation and left in a temperature controlling system at 21 degrees C for 12 days postmortem. Total RNA in lung and thoracic muscle at different time point was extracted and beta-actin mRNA expression was examined by RT-PCR. Semi-quantification analysis of the image of electrophoresis was performed to confirm the changes of beta-actin mRNA expression. RESULTS: beta-actin mRNA in lung of rats still could be detected at 12 days postmortem, but-disappeared in thoracic muscle at 8 days postmortem. CONCLUSION: The expression of beta-actin mRNA in lung and thoracic muscle could be a new parameter for estimation of PMI.

Actins↗
PubMed1992

Transforming growth factor-beta 1 (TGF-beta 1) up-regulation of collagen type II in primary cultures of rabbit articular chondrocytes (RAC) involves increased mRNA levels without affecting mRNA stability and procollagen processing.

The effect of transforming growth factor-beta 1 (TGF-beta 1) on collagen biosynthesis was investigated in confluent primary monolayer cultures of rabbit articular chondrocytes (RAC). Exposure to TGF-beta (0.1, 1, and 10 ng/ml) in serum-free medium caused a dose- and time-dependent stimulation of collagen biosynthesis associated with an increase of steady-state levels of procollagen type II mRNA. Elevation of the mRNA steady-state did not result from a stabilization of the transcript, as shown by measure of the mRNA half-life. Electrophoresis (SDS-PAGE) showed that TGF-beta stimulates the synthesis of most collagen isotypes, including type II, without qualitative change in their distribution. Moreover, pulse-chase experiments revealed that TGF-beta did not affect the processing rate of type II procollagen. TGF-beta slightly stimulated the production of prostaglandin E2 (PGE2), which could in turn exert an inhibition on collagen synthesis. However, addition of indomethacin to block prostaglandin synthesis did not further enhance the TGF-beta-induced stimulation of collagen production, suggesting that this mediator was not implicated in the effect. Moreover, TGF-beta increased steady-state levels of procollagen type II, I, and III mRNAs even in the presence of indomethacin. Despite these increased mRNA levels, only the production of type II collagen was significantly augmented, suggesting that type I procollagen mRNA was not fully translated. In addition, the TGF-beta-induced stimulation of collagen synthesis was observed whenever ascorbic acid is added or not in the culture medium. In conclusion, TGF-beta, which is present in great amount in bone and cartilage, can increase the collagen production of cultured RAC and might therefore play a role in the early events of cartilage repair, such as those observed in osteoarthritis.

Animals↗
PubMed2004

CD2-CD48 interactions promote interleukin-2 and interferon-gamma synthesis by stabilizing cytokine mRNA.

CD2-CD48 interactions enhance T cell receptor-driven mouse T lymphocyte activation. However, the mechanism is not well understood. Here we show that blockade of CD2-CD48 interactions with anti-CD48 monoclonal antibody (mAb) inhibited interleukin (IL)-2 and interferon (IFN)-gamma expression, as well as T cell proliferation in response to mitogenic anti-CD3 mAb, although more potent inhibition resulted from blocking CD28-CD80/CD86 interactions. Blockade of both CD2 and CD28 costimulation abrogated T cell proliferation and cytokine synthesis. Conversely, T cells stimulated with immobilized anti-CD3 and anti-CD2 mAb exhibited increased proliferation and IL-2 and IFN-gamma expression, although a stronger enhancing effect was obtained with immobilized anti-CD3 and anti-CD28 mAb. Concurrent CD2 and CD28 costimulation caused a further increase in proliferation and cytokine synthesis. Stimulation of purified T cells with microsphere-immobilized anti-CD3 and anti-CD2 mAb increased IL-2 and IFN-gamma mRNA stability. However, CD28 costimulation had a stronger enhancing effect on IL-2 and IFN-gamma mRNA stability that was not further increased by concomitant CD2 signaling. CD2, therefore, costimulates T cell activation by stabilizing cytokine mRNA transcripts, albeit with less efficiency than CD28.

Animals↗
PubMed2005

Induction of human spermine oxidase SMO(PAOh1) is regulated at the levels of new mRNA synthesis, mRNA stabilization and newly synthesized protein.

The oxidation of polyamines induced by antitumour polyamine analogues has been associated with tumour response to specific agents. The human spermine oxidase, SMO(PAOh1), is one enzyme that may play a direct role in the cellular response to the antitumour polyamine analogues. In the present study, the induction of SMO(PAOh1) enzyme activity by CPENSpm [N1-ethyl-N11-(cyclopropyl)methyl-4,8,diazaundecane] is demonstrated to be a result of newly synthesized mRNA and protein. Inhibition of new RNA synthesis by actinomycin D inhibits both the appearance of SMO(PAOh1) mRNA and enzyme activity. Similarly, inhibition of newly synthesized protein with cycloheximide prevents analogue-induced enzyme activity. Half-life determinations indicate that stabilization of SMO(PAOh1) protein does not play a significant role in analogue-induced activity. However, half-life experiments using actinomycin D indicate that CPENSpm treatment not only increases mRNA expression, but also leads to a significant increase in mRNA half-life (17.1 and 8.8 h for CPENSpm-treated cells and control respectively). Using reporter constructs encompassing the SMO(PAOh1) promoter region, a 30-90% increase in transcription is observed after exposure to CPENSpm. The present results are consistent with the hypothesis that analogue-induced expression of SMO(PAOh1) is a result of increased transcription and stabilization of SMO(PAOh1) mRNA, leading to increased protein production and enzyme activity. These data indicate that the major level of control of SMO(PAOh1) expression in response to polyamine analogues exposure is at the level of mRNA.

Dactinomycin↗
PubMed2006

Maintenance of CCL5 mRNA stores by post-effector and memory CD8 T cells is dependent on transcription and is coupled to increased mRNA stability.

Immunological memory is associated with the display of improved effector functions by cells of the adaptive immune system. The storage of untranslated mRNA coding for the CCL5 chemokine by CD8 memory cells is a new process supporting the immediate display of an effector function. Here, we show that, after induction during the primary response, high CCL5 mRNA levels are specifically preserved in CD8 T cells. We have investigated the mechanisms involved in the long-term maintenance of CCL5 mRNA levels by memory CD8 T cells. We demonstrate that the CCL5 mRNA half-life is increased in memory CD8 T cells and that these cells constitutively transcribe ccl5 gene. By inhibiting ccl5 transcription using IL-4, we demonstrate the essential role of transcription in the maintenance of CCL5 mRNA stores. Finally, we show that these stores are spontaneously reconstituted when the inhibitory signal is removed, indicating that the transcription of ccl5 is a default feature of memory CD8 T cells imprinted in their genetic program.

3T3 Cells↗