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PubMed1994

Coupling between mRNA synthesis and mRNA stability in Escherichia coli.

Transiently stable products derived from the endonuclease cleavage of transcripts from the secEnusG and rplKAJLrpoBC operons have been identified. Cleavage sites for RNase III occur in the leader of the secEnusG transcript and in the L12-beta intercistronic space of the rplKAJLrpoBC transcript. A single RNase E cleavage site was located in the L1-L10 intergenic space. Inactivation of RNase III and RNase E results respectively in a one- to twofold and a greater than 10-fold stabilization of five mRNA sequences from within the secE, nusG, L11-L1, L10 and beta encoding cistrons. The relative amounts of each of these five mRNA sequences were found to be nearly constant when measured either in the presence or absence of cleavage by RNase III or RNase E. This clearly implies that any increases in the stability of these mRNA sequences resulting from the inactivation of processing by RNase III or RNAase E are counterbalanced by changes in the mRNA synthesis rates. The mechanism that links mRNA synthesis to mRNA decay is not known.

Bacterial Proteins↗
PubMed1996

Progesterone together with estradiol promotes luteinizing hormone beta-subunit mRNA stability in rat pituitary cells cultured in vitro.

The present study examined the role of ovarian steroids, estradiol and/or progesterone in the regulation of luteinizing hormone beta-subunit (LH-beta) mRNA levels and LH release in the rat anterior pituitary cells cultured in vitro. When estradiol (10 nmol/l) and/or progesterone (100 nmol/l) were added to the cultures, neither estradiol or progesterone nor both together altered the basal LH-beta mRNA levels or LH release. Continuous exposure to gonadotropin-releasing hormone (GnRH, 0.2 nmol/l) for 24 h markedly induced LH-beta mRNA accumulation, and in this experimental condition, progesterone alone and progesterone + estradiol further augmented GnRH-induced LH-beta mRNA levels and LH release. Then we explored further the possibility that ovarian steroids are involved in modulating LH-beta mRNA stability in cultured rat pituitary cells where transcription was inhibited by actinomycin D. Anterior pituitary cells were preincubated with GnRH (0.2 nmol/l) for 16 h and, after removing GnRH from culture medium, the cells were incubated further in the presence of actinomycin D (5 mumol/l) for 24 h. The LH-beta mRNA levels gradually declined to about 30% of the control values (zero time point after GnRH removal) in a time-dependent manner. During this period, either progesterone alone or progesterone + estradiol clearly blocked the degradation of LH-beta mRNA species. These results indicate that ovarian steroids promote LH-beta mRNA stability, thereby contributing to the maintenance of GnRH-stimulated LH-beta mRNA levels.

Animals↗
PubMed1994

Platelet-derived growth factor B-chain gene expression in mesangial cells: effect of phorbol ester on gene transcription and mRNA stability.

We have investigated the effect of phorbol 12-myristate 13-acetate (PMA) on platelet-derived growth factor (PDGF) B-chain gene transcription as well as on mRNA stability in cultured human mesangial cells. Addition of actinomycin to cells stimulated with PMA decreases steady state levels of PDGF-B chain mRNA analysed by solution hybridization assay. PDGF-B chain gene transcription was also assayed directly by measuring elongation of transcripts in isolated nuclei followed by hybridization of labeled RNA transcripts to a cDNA encoding for PDGF-B chain. Our data show that PMA induces PDGF-B chain gene transcription by approximately 2-fold. alpha-Amanitin, an RNA polymerase II inhibitor, blocked transcription by more than 70%. In addition, we determined the effect of PMA on the halflife of PDGF-B chain mRNA directly by pulse chase method. In human mesangial cells, the PDGF-B chain mRNA exhibited halflife of approximately 105 min. In the presence of PMA, the halflife of PDGF-B chain mRNA was reduced to approximately 72 min. These studies indicate that regulation of PDGF-B chain gene by PMA in human mesangial cells involves a coordinate effort at the level of transcription and mRNA stability.

Amanitins↗
PubMed2000

HuR regulates p21 mRNA stabilization by UV light.

Expression of the cyclin-dependent kinase inhibitor p21 is highly induced by many stresses, including exposure to short-wavelength UV light (UVC), which increases p21 mRNA stability. Investigation into the mechanisms underlying this stabilization process revealed that proteins present in cytoplasmic lysates of human RKO colorectal carcinoma cells formed complexes with p21 mRNA that were inducible by treatment with UVC and other stress agents. The ubiquitous Elav-type RNA-binding protein HuR was identified within the p21 mRNA-protein complexes, as antibodies recognizing HuR supershifted these complexes and revealed HuR-immunoreactive proteins complexing with p21 mRNA on Western blots. Lowering of endogenous HuR levels through expression of antisense HuR decreased p21 RNA-protein complexes, greatly reduced the UVC inducibility and half-life of p21 mRNA, and prevented UVC-mediated induction of luciferase activity in p21 3' untranslated region-containing reporter constructs. Our findings indicate that HuR plays a major role in regulating stress-induced p21 expression by enhancing p21 mRNA stability and that these effects are coupled to HuR's elevated presence in the cytoplasm.

Antigens, Surface↗
PubMed2002

Correlation between intrinsic mRNA stability and the affinity of AUF1 (hnRNP D) and HuR for A+U-rich mRNAs.

Presence of A+U-rich elements (AREs) within 3'-untranslated regions (3'UTRs) of numerous mRNAs has been associated with rapid mRNA turnover; however, the interaction of specific factors with AREs is also associated with mRNA stabilization. Recently, two ARE binding proteins with putative mRNA destabilizing (AUF1) and stabilizing (HuR) properties have been described. However, no direct comparison ofAUF1 and HuR binding properties has been made. Therefore, we examined the relative affinities of p37AUF1 and HuR for a diverse set ofARE-containing mRNAs encoding beta-adrenergic receptors, a proto-oncogene, and a cytokine. We find that high-affinity AUF1 binding appears to require elements beyond primary nucleotide sequence. In contrast, binding of HuR appears considerably less constrained. As a functional correlate, we determined the ability of these specific mRNA sequences to affect the stability of chimeric beta-globin mRNA constructs. Although the relative affinity ofAUF1 and HuR are generally predictive of mRNA stability, we find that certain mRNA sequences do not conform to these generalizations.

3' Untranslated Regions↗
PubMed1995

Hypoxic induction of vascular endothelial growth factor (VEGF) in human epithelial cells is mediated by increases in mRNA stability.

Vessel growth is often associated with ischemia. VEGF, a potent angiogenic factor, has been shown to be induced by low oxygen concentrations. These studies were conducted to investigate the molecular basis of the hypoxia-induced increase in VEGF mRNA. Run-on analysis of VEGF revealed a minimal increase in the rate of gene transcription in a human retinal epithelial cell line grown under hypoxic conditions. Examination of VEGF mRNA stability revealed that the half-life of VEGF transcripts under normoxia was short, 30-45 min, but was dramatically increased to 6-8 h in cells grown under hypoxia. Cobalt chloride, which elevates VEGF and has been suggested to be similar to hypoxia in its mechanism of action, had only a slight effect on decay rate. We postulate that hypoxia-induced increases in mRNA stability provide the sustained increases in VEGF mRNA levels necessary to support a neovascular response.

Cells, Cultured↗
PubMed2003

Mitochondrial mRNA stability and polyadenylation during anoxia-induced quiescence in the brine shrimp Artemia franciscana.

Polyadenylation of messenger RNA is known to be an important mechanism for regulating mRNA stability in a variety of systems, including bacteria, chloroplasts and plant mitochondria. By comparison, little is known about the role played by polyadenylation in animal mitochondrial gene expression. We have used embryos of the brine shrimp Artemia franciscana to test hypotheses regarding message stability and polyadenylation under conditions simulating anoxia-induced quiescence. In response to anoxia, these embryos undergo a profound and acute metabolic downregulation, characterized by a steep drop in intracellular pH (pH(i)) and ATP levels. Using dot blots of total mitochondrial RNA, we show that during in organello incubations both O(2) deprivation and acidic pH (pH 6.4) elicit increases in half-lives of selected mitochondrial transcripts on the order of five- to tenfold or more, relative to normoxic controls at pH 7.8. Polyadenylation of these transcripts was measured under the same incubation conditions using a reverse transcriptase-polymerase chain reaction (RT-PCR)-based assay. The results demonstrate that low pH and anoxia promote significant deadenylation of the stabilized transcripts in several cases, measured either as change over time in the amount of polyadenylation within a given size class of poly(A)(+) tail, or as the total amount of polyadenylation at the endpoint of the incubation. This study is the first direct demonstration that for a metazoan mitochondrion, polyadenylation is associated with destabilized mRNA. This pattern has also been demonstrated in bacteria, chloroplasts and plant mitochondria and may indicate a conserved mechanism for regulating message half-life that differs from the paradigm for eukaryotic cytoplasm, where increased mRNA stability is associated with polyadenylation.

Adenosine Triphosphate↗
PubMed1995

Pentobarbital-induced changes in Drosophila glutathione S-transferase D21 mRNA stability.

The Drosophila glutathione S-transferase (gstD) genes are a family of divergently transcribed, intronless genes and pseudogenes. Under control conditions, the steady-state level of gstD1 mRNA is 20-fold higher than that of the gstD21 mRNA despite a lower transcription rate of the gstD1 gene. The GST D1 protein level is four times as abundant as the GST D21 protein. The gstD1 and gstD21 genes responded rapidly to pentobarbital (PB) as changes in mRNA levels were detectable within 30 min of treatment. Maximal induction of gstD1 and gstD21 resulted in 3-fold and 20-fold elevation of their respective mRNA levels. The major mechanism for the increase in gstD1 mRNAs appears to be transcriptional activation. The 2-fold increase in the rate of gstD21 transcription, however, cannot fully account for the 20-fold increase in the steady-state level of gstD21 mRNA. Therefore, post-transcriptional mechanism(s) should also be responsible for the increase of gstD21 mRNA by PB. Because the gstD21 mRNA is relatively unstable under control conditions, induction of the intronless gstD21 mRNA by PB occurs mainly at the level of enhanced mRNA stability. The GST D1 protein level in adult Drosophila was increased approximately 2-fold after PB treatment, whereas the GST D21 level remained relatively the same. Thus, an increase in gstD21 mRNA stability by PB treatment is probably coupled to a regulatory effect at the translational level.

Animals↗
PubMed1996

Differential mRNA decay within the transfer operon of plasmid R1: identification and analysis of an intracistronic mRNA stabilizer.

Processing of the transfer operon mRNA of the conjugative resistance plasmid R1-19 results in the accumulation of stable traA mRNAs. The stable traA transcripts found in vivo have identical 3' ends within downstream traL sequences, but vary at their 5' ends. The 3' ends determined coincide with the 3' base of a predicted large clover-leaf-like RNA secondary structure. Here we demonstrate that this putative RNA structure, although part of a coding sequences, stabilizes the upstream traA mRNA very efficiently. We also show that the 3' ends of the stable mRNAs are formed posttranscriptionally and not by transcription termination. Half-life determinations reveal the same half-lives of 13 +/- 2 min for the traA mRNAs transcribed from hybrid lac-traAL-cat test plasmids, the R1-19 plasmid, and the F plasmid. Protein expression experiments demonstrate that the processed stable traA mRNA is translationally active. Partial deletions of sequences corresponding to the predicted secondary structure within the traL coding region drastically reduce the chemical and functional half-life of the traA mRNA. The results presented here unambiguously demonstrate that the proposed secondary structure acts as an efficient intracistronic mRNA stabilizer.

Bacterial Outer Membrane Proteins↗
PubMed1997

Promoter-independent cold-shock induction of cspA and its derepression at 37 degrees C by mRNA stabilization.

The gene for CspA, the major cold-shock protein of Escherichia coli is known to be dramatically induced upon temperature downshift. Here, we report that three-base substitutions around the Shine-Dalgarno sequence in the 159-base 5'-untranslated region of the cspA mRNA stabilizes the mRNA 150-fold, resulting in constitutive expression of cspA at 37 degrees C. This stabilization was found to be at least partially due to resistance against RNase E degradation. The cold-shock induction of cspA was also achieved by exchanging its promoter with the non-cold-shock Ipp promoter. The results presented indicate that the cspA gene is efficiently transcribed even at 37 degrees C. However, the translation of the cspA mRNA is blocked because of its extreme instability at 37 degrees C. The presented results also demonstrate that the cspA gene is constitutively transcribed at all temperatures; however, its expression at 37 degrees C is prevented by destabilizing its mRNA.

Bacterial Proteins↗
PubMed1996

Differential contributions of two elements of rho-independent terminator to transcription termination and mRNA stabilization.

The hallmark features of rho-independent transcription terminators are a G(+)C-rich dyad symmetry sequence followed by a run of T residues on a sense strand. Both of these structural elements are required for efficient transcription termination. Besides its primary function, rho-independent terminators are also known to enhance expression of an upstream gene by stabilizing RNA in a few cases. The Escherichia coli crp gene encoding cAMP receptor protein (CRP) contains a typical rho-independent terminator. To gain further insight into the roles of the G(+)C-rich dyad symmetry sequence and the poly(T) tract both in transcription termination and mRNA stabilization, we constructed a series of variant crp terminators and analyzed their abilities regarding these two functions. Disruption of the G(+)C-rich dyad symmetry sequence almost completely eliminated terminator activity while disruption of the poly(T) tract reduced terminator activity significantly but not completely. Thus, the contribution of the G(+)C-rich dyad symmetry sequence to transcription termination is larger than that of the poly(T) tract. Disruption of the G(+)C-rich dyad symmetry region reduced expression of the upstream crp gene by accelerating the rate of mRNA degradation. However, disruption of the poly(T) sequence had no effect on the stability of the crp mRNA, indicating that the poly(T) tract plays no role in mRNA stabilization. When the crp terminator was replaced by terminators derived from other genes, the fusion genes expressed the crp mRNA at the same level as did the native crp gene, suggesting that the mRNA stabilization effect is probably a general nature of rho-independent terminators.

Bacterial Proteins↗
PubMed2003

P-glycoprotein (MDR1) expression in leukemic cells is regulated at two distinct steps, mRNA stabilization and translational initiation.

Multidrug resistance in acute myeloid leukemia is often conferred by overexpression of P-glycoprotein, encoded by the MDR1 gene. We have characterized the key regulatory steps in the development of multidrug resistance in K562 myelogenous leukemic cells. Unexpectedly, up-regulation of MDR1 levels was not due to transcriptional activation but was achieved at two distinct post-transcriptional steps, mRNA turnover and translational regulation. The short-lived (half-life 1 h) MDR1 mRNA of naive cells (not exposed to drugs) was stabilized (half-life greater than 10 h) following short-term drug exposure. However, this stabilized mRNA was not associated with translating polyribosomes and did not direct P-glycoprotein synthesis. Selection for drug resistance, by long-term exposure to drug, led to resistant lines in which the translational block was overcome such that the stabilized mRNA was translated and P-glycoprotein expressed. The absence of a correlation between steady-state MDR1 mRNA and P-glycoprotein levels was not restricted to K562 cells but was found in other lymphoid cell lines. These findings have implications for the avoidance or reversal of multidrug resistance in the clinic.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1986

Posttranscriptional control of Klebsiella pneumoniae nif mRNA stability by the nifL product.

Posttranscriptional control of nif mRNA stability was demonstrated by functional and chemical analyses, using specific probes for four nif transcripts. In the wild type, nif transcripts (except nifLA) were stable during derepression, with half-lives of approximately 30 min. They were dramatically destabilized by O2 or elevated temperature (41 degrees C) and to a lesser extent by NH4+. In contrast, the nifLA message was not particularly stable, and posttranscriptional control was not evident. In NifL- strains, both forms of analysis indicated that the nifL product was involved in nif mRNA destabilization in the presence of O2 and NH4+.

Acetates↗
PubMed2005

Down-regulation of Na+/Ca2+ exchanger by fluvastatin in rat cardiomyoblast H9c2 cells: involvement of RhoB in Na+/Ca2+ exchanger mRNA stability.

We investigated the effect of fluvastatin (Flv), an HMG-CoA reductase inhibitor, on Na(+)/Ca(2+) exchanger 1 (NCX1) expression in H9c2 cardiomyoblasts. Reverse transcriptase-polymerase chain reaction analyses revealed that Flv decreased NCX1 mRNA in a concentration- and time-dependent manner and NCX1 protein. This effect of Flv was caused by the inhibition of HMG-CoA reductase, because Flv did not affect the NCX1 mRNA in the presence of mevalonate. Flv-induced down-regulation of NCX1 mRNA was also cancelled by farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), suggesting an involvement of small G-proteins. However, overexpression of neither constitutive active RhoA nor Ras affected NCX1 mRNA. In contrast, intracellular expression of C3 toxin, a specific inhibitor of Rho family proteins, decreased NCX1 mRNA, suggesting that Flv decreases NCX1 mRNA by inhibiting a signaling pathway of Rho family proteins other than RhoA. On the other hand, lysophosphatidylcholine (LPC), an activator of Rho signaling, increased both NCX1 mRNA and protein in a C3 toxin-sensitive manner. Western blot analyses revealed that membrane-associated RhoB, which is isoprenylated by either FPP or GGPP, was decreased by Flv but was increased by LPC. Selective inhibition of gene expression by short interfering RNA duplex showed that RhoB but not RhoA is involved in the regulation of NCX1 mRNA and protein. When transcription was blocked by 5,6-dichlorobenzimidazole riboside, the NCX1 mRNA stability was decreased by Flv. Long-term treatment of rat with Flv in vivo also down-regulated the cardiac NCX1 mRNA. These results suggest that a RhoB-mediated signaling pathway regulates cardiac NCX1 levels by controlling the NCX1 mRNA stability.

Animals↗
PubMed1999

p38 mitogen-activated protein kinase regulates cyclooxygenase-2 mRNA stability and transcription in lipopolysaccharide-treated human monocytes.

p38 mitogen-activated protein kinase (MAPK) is activated by inflammatory stimuli such as bacterial lipopolysaccharide (LPS), interleukin-1, and tumor necrosis factor. We have previously shown that the pyridinyl imidazole SB 203580, which inhibits it, blocks the interleukin-1 induction of cyclooxygenase-2 (COX-2) and matrix metalloproteinase 1 and 3 mRNAs in fibroblasts. Here we explore the role of p38 MAPK in the response of human monocytes to LPS. 0.1 microM SB 203580 significantly inhibited the LPS induction of COX-2 and tumor necrosis factor protein and mRNAs. The activity of MAPK-activated protein kinase-2 (a substrate of p38 MAPK) in the cells was commensurately reduced. Some isoforms of c-jun N-terminal kinase (which is also activated by LPS) are sensitive to SB 203580; the inhibitor had little effect on monocyte c-jun N-terminal kinases up to 2 microM. We investigated the mechanism of inhibition of COX-2 induction. Transcription (measured by a nuclear run-on assay) was 60% inhibited by SB 203580 (2 microM). Importantly, we found that p38 MAPK was essential for stabilizing COX-2 mRNA: when cells stimulated for 4 h with LPS were treated with actinomycin D, COX-2 mRNA decayed slowly. Treatment of stimulated cells with 2 microM SB 203580 caused a rapid disappearance of COX-2 mRNA, even with actinomycin D present. We conclude p38 MAPK plays a role in the transcription and stabilization of COX-2 mRNA.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1996

Control of mRNA stability in higher plants.

The degradation rates of different mRNAs in higher plants can vary over a broad range and are regulated by a variety of endogenous and exogenous stimuli. During the past several years, efforts to better understand the control of mRNA stability in plants have increased considerably and this has led to improved methodologies and important mechanistic insights. In this review, we highlight some of the most interesting examples of plant transcripts that are controlled at the level of mRNA decay and discuss what has been learned from their study. Experiments that implicate or demonstrate the involvement of particular cis- and trans-acting factors in mRNA decay pathways are a major focus, as are those experiments that have led to mechanistic models. Emphasis is also placed on studies that address the relationship between translation and mRNA stability. Our current knowledge indicates that some of the determinants and pathways for mRNA decay may differ in plants compared to other eukaryotes, whereas others appear to be similar. This knowledge, coupled with the availability of biochemical, molecular and genetic approaches to elucidate plant mRNA decay mechanisms, should continue to lead to findings of novel and general significance.

Base Sequence↗
PubMed2001

Increased bar minigene mRNA stability during cell growth inhibition.

Bacteriophage lambda is unable to grow vegetatively on Escherichia coli mutants defective in peptidyl-tRNA hydrolase (Pth) activity. Mutations which allow phage growth on the defective host have been located at regions named bar in the lambda genome. Expression of wild-type bar regions from plasmid constructs results in inhibition of protein synthesis and lethality to Pth-defective cells. Two of these wild-type bar regions, barI+ and barII+, contain minigenes with similar AUG-AUA-stop codon sequences preceded by different Shine-Dalgarno (SD) and spacer regions. The induced expression of barI+ and barII+ regions from plasmid constructs resulted in similar patterns of protein synthesis inhibition and cell growth arrest. Therefore, these deleterious effects may stem from translation of the transcripts containing the minigene two-codon 'ORF' (open reading frame). To test for this possibility, we assayed the effect of point mutations within the barI minigene. The results showed that a base pair substitution within the SD and the two-codon 'ORF' sequences affected protein synthesis and cell growth inhibition. In addition, mRNA stability was altered in each mutant. Higher mRNA stability correlated with the more toxic minigenes. We argue that this effect may be caused by ribosome protection of the mRNA in paused complexes as a result of deficiency of specific tRNA.

Bacterial Proteins↗
PubMed2005

Multidrug resistance polypeptide 1 (MDR1, ABCB1) variant 3435C>T affects mRNA stability.

OBJECTIVES: ABCB1 (multidrug resistance 1 polypeptide, MDR1, Pgp) is a multispecific efflux transporter of drugs and xenobiotics. Among numerous polymorphisms in human ABCB1, the synonymous SNP 3435C > T has been associated with decreased mRNA and protein levels, via unknown mechanisms. METHODS: To search for cis-acting polymorphism affecting transcription or mRNA processing, we used 3435C > T as a marker single nucleotide polymorphism (SNP), for measuring differences in allelic mRNA expression. Ratios of allelic abundance in genomic DNA and mRNA (after conversion to cDNA) were measured quantitatively with a primer extension assay, in human liver samples. RESULTS: mRNA expression of the 3435C allele was significantly higher than that of the 3435T allele (3435C/3435T ratios ranging from 1.06-1.61). Cotransfection of equal amounts of ABCB1 expression plasmids containing 3435C or 3435T also revealed higher 3435C mRNA expression. Increasing 3435C/3435T ratios after cessation of transcription indicated that the 3435C > T substitution decreases mRNA stability. 3435C > T is in strong linkage disequilibrium with two other coding SNPs (1236C > T and 2677G > T) forming two abundant haplotypes (ABCB1*1 and ABCB1*13). Transfection of all possible combinations of these three SNPs demonstrated that only 3435T is associated with lower mRNA levels. Calculations of mRNA folding, using Mfold, suggested an effect on mRNA secondary structure. CONCLUSIONS: the abundant 3435C > T SNP appears to be a main factor in allelic variation of ABCB1 mRNA expression in the liver, by changing mRNA stability.

ATP-Binding Cassette Transporters↗