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PubMed2006

Stability of CXCL-8 and related AU-rich mRNAs in the context of hepatitis C virus replication in vitro.

BACKGROUND: Previous studies have shown that levels of CXCL-8 are elevated in patients with chronic hepatitis C virus (HCV) infection and are highest in nonresponders to interferon (IFN) therapy and that CXCL-8 inhibits IFN antiviral action. CXCL-8 expression involves AU-rich elements (AREs) in 3' untranslated regions that regulate mRNA stability. CXCL-8 mRNA stability was, therefore, investigated in the context of HCV replication in 4 replicon cell lines, Huh7 and Huh7.5 cells, and primary human fetal hepatocytes. METHODS: The half-life of CXCL-8 mRNA was measured by use of real-time reverse-transcription polymerase chain reaction following tumor necrosis factor (TNF)- alpha induction in the presence and absence of inhibitors of transcription and translation. Cellular mRNAs containing AREs were assessed by custom microarray analyses. RESULTS: The half-life of CXCL-8 mRNA increased in 3 of 4 HCV replicon cell lines, particularly after treatment with TNF- alpha , a potent inducer of CXCL-8. CXCL-8 mRNA was superinduced by TNF- alpha in the presence of the protein-synthesis inhibitor cycloheximide. Analysis of >1500 ARE-containing cellular mRNAs, by use of microarrays, revealed that CXCL-8 and other newly identified ARE genes were induced by TNF- alpha in Huh7 cells and were coordinately regulated. CONCLUSION: The data suggest that increased CXCL-8 gene expression in the context of HCV replication involves posttranscriptional events.

Adenine↗
PubMed1998

The phosphorylation of protein S6 modulates the interaction of the 40 S ribosomal subunit with the 5'-untranslated region of a dictyostelium pre-spore-specific mRNA and controls its stability.

AC914 mRNA, a pre-spore-specific mRNA that accumulates only in the post-aggregation stage of development, is transcribed constitutively as shown by nuclear run-off experiments and by fusing its promoter to the luciferase reporter gene. The same mRNA disappears quickly from disaggregated cells. If the 5'-untranslated region (5'UTR) of the constitutively expressed Actin 15 mRNA is substituted for the 5'UTR of AC914 mRNA, this can no longer be destabilized and accumulates both in growing and disaggregated cells. If the 5'UTR of AC914 mRNA is substituted for the 5'UTR of Actin 15 mRNA, the latter accumulates only in aggregated cells. Pactamycin, but not other inhibitors of protein synthesis, prevents AC914 mRNA from being destabilized in disaggregated cells, suggesting a role of 40 S subunits in the destabilization. This has been confirmed by using an in vitro system in which the in vivo stability of different mRNAs is reproduced. A protein kinase A-dependent phosphorylation of ribosomal protein S6 determines whether 40 S subunits are capable or not of destabilizing AC914 mRNA in the in vitro system.

5' Untranslated Regions↗
PubMed1999

Calcium ionophore upregulation of AUUUA-specific binding protein activity is contemporaneous with granulocyte macrophage colony-stimulating factor messenger RNA stabilization in AML14.3D10 cells.

Eosinophils produce granulocyte macrophage colony-stimulating factor (GM-CSF), which enhances their survival and function. In T cells and fibroblasts, GM-CSF production is controlled predominantly by variable messenger RNA (mRNA) stability involving 3' untranslated region (3' UTR) adenosine-uridine-rich elements (AREs) and sequence-specific mRNA binding proteins. However, the mode of regulation of this critical cytokine remains unknown in eosinophils. Therefore, we measured GM-CSF mRNA decay in an eosinophil-like cell line (AML14.3D10) and, with a radiolabeled GM-CSF RNA probe, asked whether ARE-specific, mRNA binding proteins were present in cytoplasmic lysates of these cells. Human GM-CSF mRNA transfected into unstimulated AML14.3D10 cells decayed with a half-life of 6 min, which increased to 14 min after 1 h, and to 22 min after 2 h, of ionophore-mediated activation. GM-CSF RNA mobility shift assays using cytoplasmic extracts from resting or ionophore-stimulated AML14.3D10 cells revealed multiple RNA-protein complexes of 55, 60, 85, 100, and 125 kD. A 47-kD complex was also detected with an 80-base RNA probe containing four consecutive AUUUA motifs. On the basis of competition studies, all of the observed binding protein activities interacted with the 3' UTR AREs. In addition, binding activity increased 2.5-fold in cytoplasmic lysates from cells stimulated with calcium ionophore for 2 h, contemporaneous with GM-CSF mRNA stabilization. These data provide direct evidence that ionophore stabilizes GM-CSF mRNA in AML14.3D10 cells and simultaneously increases the activity of a series of AUUUA-specific mRNA binding proteins. We conclude that the interaction of AU-specific binding proteins may stabilize GM-CSF mRNA in activated eosinophil-like cell lines.

3' Untranslated Regions↗
PubMed1991

Hybrid molecules: insights into plasminogen activator function.

Hybrid molecules containing the catalytic domain of either tissue plasminogen activator (tPA) or single chain urokinase-type plasminogen activator (scuPA), and the fibrin binding domain of a murine antifibrin monoclonal antibody were constructed using either cDNA or genomic DNA encoding the plasminogen activator and genomic DNA encoding antifibrin monoclonal antibody 59D8. In order to optimize expression of these fusion proteins in hybridoma cells, we compared plasminogen activator 3' UT domains (which decrease mRNA stability) with immunoglobulin and beta globin 3' UT domains (which increase mRNA stability). The presence of the plasminogen activator 3' UT domain resulted in approximately tenfold lower steady-state mRNA levels, and 300 to 500-fold lower levels of expressed functional protein. The initial goal of these studies was to increase the fibrinolytic potency and selectivity of tPA or scuPA. Fusion proteins comprising an antifibrin antibody domain and the catalytic domain of either tPA or scuPA were expressed and shown to have very different properties. The fusion protein that comprised the Fab portion of an antifibrin antibody and the catalytic domain of tPA, while displaying antigen binding properties indistinguishable from those of the parent antibody and amidolytic activity similar to that of tPA, was not more efficient than tPA in an in vitro clot lysis assay. In contrast, it had been shown that tPA chemically coupled to the same antibody was four- to sixfold more efficient in fibrinolysis both in vitro and in vivo. A recombinant scuPA-antifibrin antibody hybrid, however, was sixfold more potent than scuPA in vitro and 20-fold more potent in a rabbit thrombolysis model. An explanation for this apparent discrepancy may relate to the requirement for stimulation by fibrin in order for tPA to achieve its maximal catalytic activity, a property that was demonstrated to have been lost in the antifibrin-tPA fusion protein. In contrast, the activity of urokinase is independent of the presence of fibrin. This may explain the greater success achieved in enhancing catalytic activity in the urokinase-antifibrin fusion protein. It is of additional interest that fibrin or soluble fibrin fragments stimulate the catalytic activity of both tPA and the isolated tPA B chain, demonstrating that at least part of the enhanced catalytic activity of tPA observed in the presence of fibrin is independent of fibrin binding either by the tPA kringles or finger domain (or any heavy chain domain). These data indicate that it is possible to construct recombinant hybrid molecules in which both plasminogen activator catalytic function and antibody binding are preserved.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed2002

Inhibition of estrogenic stimulation of gene expression by genistein.

Two principle soy-derived isoflavones, genistein and daidzein, are believed to play a key role in inhibiting tumor growth. The molecular basis of the anti-tumor activity of these two isoflavones has not been fully established. To determine the mechanism of action of the above phytochemicals on estrogen-responsive genes, we tested the effect of the same on the expression of Estrogen-Regulated mRNA Stabilizing Factor (E-RmRNASF). E-RmRNASF is expressed in the liver in response to estrogen, and is responsible for estrogen-mediated stabilization of apolipoprotein II mRNA (Ratnasabapathy, 1995, Cell. Mol. Biol. Res, 41: 583-594). The estrogen-mediated hepatic expression of apolipoprotein II mRNA is regulated transcriptionally, and also post-transcriptionally in part by stabilization of its mRNA. E-RmRNASF protects the RNA from targeted endonucleolytic degradation. The hepatic expression of E-RmRNASF is also modulated by certain estrogenic and antiestrogenic nonsteroidal environmental xenobiotics (Ratnasabapathy et al., 1997, Biochem. Pharmacol., 53: 1425-1434). Roosters were administered estrogen, genistein, or daidzein parenterally and tested for hepatic expression of E-RmRNASF. Expression of E-RmRNASF in the livers was stimulated in roosters who received estrogen and genistein, indicating that they are agonistic at the chicken estrogen receptor. However, a lack of induction of E-RmRNASF expression in the liver was seen with control roosters treated with the vehicle and those treated with daidzein. To determine whether daidzein was anti-estrogenic, roosters were given combinations of estrogen and increasing concentrations of daidzein. Daidzein at concentrations ranging from 5-1000 micromole/kg failed to antagonize stimulation of E-RmRNASF by 5 micromoles/kg estrogen. To determine whether genistein or daidzein exerted partial agonistic effects, roosters were given increasing concentrations of genistein, daidzein or estrogen alone, or combinations of estrogen and increasing doses of genistein or daidzein. At low to intermediate concentrations genistein by itself failed to stimulate E-RmRNASF, and was agonistic only at high concentrations. Genistein at the low concentrations failed to antagonize estrogenic stimulation of E-RmRNASF. At the intermediate concentrations however, genistein blocked stimulation of E-RmRNASF by estrogen, even though by itself could not exert a stimulatory effect. At the higher concentrations genistein stimulated E-RmRNASF regardless of the presence or absence of estrogen. At the higher ratios, the lack of inhibition of estrogenic stimulation by genistein was most likely due to its own agonistic activity. Therefore, genistein appears to behave as a partial agonist; behaves as an agonist by itself, and as an antagonist in the presence of estrogen. However, daidzein did not display any estrogenic or antiestrogenic activity at the concentrations tested.

Animals↗
PubMed1995

Thyroid hormones stabilize acetylcholinesterase mRNA in neuro-2A cells that overexpress the beta 1 thyroid receptor.

We investigated the intracellular events involved in the 3,3',5-triiodo-L-thyronine (T3)-induced accumulation in acetylcholinesterase (AChE) activity in neuroblastoma cells (neuro-2a) that overexpress the human thyroid receptor beta 1 (hTR beta 1). Treatment of these cells with T3 increased AChE activity and its mRNAs after a lag period of 24-48 h, and these levels increased through stabilization of the transcripts by T3. T3 had no effect on the transcriptional rate or processing of AChE transcripts. The protein kinase inhibitor H7 inhibited T3-induced accumulation in AChE activity and its mRNAs, whereas okadaic acid (a potent inhibitor of phosphatases 1 and 2A) potentiated the effect of T3. Okadaic acid and H7 have no effect on the binding of hTR beta 1 to T3 or the transcriptional rate of the AChE gene. Finally, treatment of cells with T3 stimulated cytosolic serine/threonine, but not tyrosine kinase, activities. The time course analysis reveals that the increase in serine/threonine activity precedes the effect of T3 on AChE mRNAs. These results suggest that activation of a serine/threonine protein kinase pathway might be a link between nuclear thyroid hormone receptor activation and stabilization of AChE mRNA.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2002

Up-regulation of cyclooxygenase-2 expression and prostaglandin synthesis in endometrial stromal cells by malignant endometrial epithelial cells. A paracrine effect mediated by prostaglandin E2 and nuclear factor-kappa B.

We investigated the regulation of prostaglandin production in normal endometrial stromal cells (ESC) by malignant endometrial epithelial cells. We found that cyclooxygenase (COX)-2 mRNA and protein levels and prostaglandin (PG)E(2) production in ESC were significantly increased by Ishikawa malignant endometrial epithelial cell conditioned medium (MECM). By using transient transfection assays, we found that the -360/-218-bp region of the COX-2 promoter gene was critical for MECM induction of promoter activity. This MECM-responsive region contained a variant nuclear factor (NF)-kappa B site at -222 to -213 that, when mutated, completely abolished COX-2 promoter activation by MECM. Employing electrophoretic mobility shift assays, we further demonstrated that binding of NF-kappa B p65 to this NF-kappa B-binding site is, in part, responsible for the COX-2 promoter activation by MECM. To investigate further the potential effects of MECM on COX-2 mRNA stability, ESC were treated with MECM in the absence or presence of actinomycin D, a general transcription inhibitor. We found that MECM significantly increased COX-2 mRNA stability. Intriguingly, we found that PGE(2) was one of the major factors in MECM, which was responsible for up-regulating COX-2 expression in ESC. ECC-1 and HEC-1A malignant endometrial epithelial cell lines also produced significantly increased quantities of PGE(2). In conclusion, malignant endometrial epithelial cells secrete PGE(2) that induces COX-2 expression in normal endometrial stromal cells in a paracrine fashion through activation of transcription and stabilization of COX-2 mRNA.

Cyclooxygenase 2↗
PubMed1992

Proteins associated with the messenger ribonucleoprotein particle for the estrogen-regulated apolipoprotein II mRNA.

The stability of the mRNA for apolipoprotein (apo) II is regulated by estrogen [Gordon et al. (1988) J. Biol. Chem. 263, 2625-2631]. On the hypothesis tha estrogen regulation of apoII mRNA stability is mediated through mRNA-protein interaction, we have examined the messenger ribonucleoprotein particle (mRNP) for apoII mRNA following release from chicken liver polyribosomes. Polyribosomes containing undegraded apoII mRNA were obtained when tissue was homogenized without detergent, and polyribosomes were isolated following simultaneous addition of detergent and magnesium to a 20000g supernatant. ApoII mRNP released by EDTA sedimented at 12-18 S in sucrose gradients, and banded at rho = 1.4 g/mL in CsCl isopycnic centrifugation, indicative of a 3:1 ratio of protein to mRNA. A fraction in which apoII mRNP was enriched to 40-50% of total mRNP was prepared by successive size fractionation steps on sucrose gradients. Proteins associated with sucrose gradient enriched apoII mRNP were examined by iodination of UV-cross-linked proteins followed by SDS-polyacrylamide gel electrophoresis. Comparisons of proteins in highly enriched apoII mRNP to proteins in mRNP from non-estrogen-treated rooster liver did not reveal any differences. This result suggests that the major proteins associated with apoII mRNA are mRNP proteins also associated with the bulk of liver mRNAs.

Animals↗
PubMed2004

A mechanism of induction of the mouse zinc-fingers and homeoboxes 1 (ZHX1) gene expression by interleukin-2.

The effect of IL-2 on the expression of the mouse zinc-fingers and homeoboxes 1 (ZHX1) gene was investigated in a mouse cytotoxic T cell line, CTLL-2 cells. IL-2 specifically induced the expression of ZHX1 mRNA. The level of ZHX1 mRNA was decreased in the absence of IL-2. These alterations were in parallel with the status of cell proliferation. The signaling pathways involved in the induction were examined. AG-490, wortmannin, and LY294002 blocked the induction by IL-2. Nuclear run-on assays and a mRNA stability analysis revealed that the half-life of ZHX1 mRNA but not the transcription rate of the gene was increased by IL-2. Thus, we conclude that IL-2 induces the expression of the mouse ZHX1 gene in CTLL-2 cells, that both Janus kinase 3/signal transducer and activator of transcription 5 and phosphoinositide 3-kinase pathways are involved in the induction, and that the increased mRNA stability results in the induction.

Animals↗
PubMed2004

Controlling the release and production of renin.

The renin-angiotensin system (RAS) plays a pivotal role for a variety of cardiovascular functions. The diversity of renin actions is reflected by its complex control. The major stimulus for the release of renin from the vesicles in juxtaglomerular cells is determined by stretch, as induced by changes in arterial pressure. The production of renin underlies a vastly complex control network, which takes place at different levels, such as transcription and translation. With regard to transcription, important regions for binding transcription factors have been identified several years ago, but the conservation of nucleotide sequences throughout different species suggests that there might be additional binding regions of importance. At the post-transcriptional level, the modulation of renin mRNA stability is seems pivotal. The half-life of renin mRNA appears to be controlled by the interaction between several regulatory proteins, most of which are well known in other systems. Moreover, in addition to the modulation of mRNA stability, the translation efficiency seems to play a key role in determining the amount of renin to be produced.

Humans↗
PubMed2026

Inactivation of CDK12 Enhances Mitochondrial Efficiency to Suppress DNA Damage.

Inactivation of cyclin-dependent kinase 12 (CDK12) characterizes a subset of prostate cancers but it is not understood how cells adapt to declining activity of this major transcription elongation kinase. To probe this response, we developed a cell line resistant to an inhibitor targeting CDK12 and its paralog, CDK13. CDK13 can compensate for the loss of CDK12, which is why we used the dual inhibitor THZ531. Targeted drug screening of the parental and resistant cell lines revealed cross-resistance to other transcriptional kinases but no clear acquired point of vulnerability. Using genome-wide mapping of mRNA-stabilization based on metabolic labelling of RNA, we report selective mRNA stabilization of factors promoting oxidative phosphorylation in the resistant cells. We go on to show that loss of CDK12 activity enhances ATP production both in cell line models and in patient tumours. Finally, we show that dual inhibition of CDK12/13 results in excessive phosphorylation of the DNA damage H2AX in prostate cancer cells but not in our CDK12/13 inhibitor-resistant model system. In brief, we propose that inactivation of CDK12 rewires cellular energy metabolism to suppress DNA damage.

Humans↗
PubMed2002

Basic fibroblast growth factor upregulates cyclooxygenase-2 in I407 cells through p38 MAP kinase.

The intestinal cell line I407 responds to basic fibroblast growth factor (bFGF) by upregulating cyclooxygenase-2 (COX-2) mRNA and protein expression and increasing PGE(2) production. bFGF treatment of I407 cells results in phosphorylation of p38, and the p38 inhibitor SB-203580 abrogates bFGF-induced PGE(2) synthesis. Wild-type p38alpha (p38alphaWT) and dominant-negative p38alpha (p38alphaDN) stable transfectant clones of I407 cells were used to examine the role of the p38 MAP kinase pathway in the events controlling PGE(2) synthesis after treatment with bFGF. Treatment of p38alphaWT clones with bFGF resulted in increased COX-2 protein levels and PGE(2) synthesis similar to those seen in bFGF-treated control-transfected cells. In contrast, the p38alphaDN clones failed to upregulate COX-2 protein or increase PGE(2) synthesis when treated with bFGF. Exogenous arachidonate did not restore PGE(2) synthesis by p38alphaDN cells. bFGF treatment increased COX-2 mRNA stability, and the p38 inhibitor SB-203580 attenuated COX-2 mRNA stability in bFGF-treated I407 cells. These data demonstrate a crucial role for p38alpha in growth factor-induced PGE(2) synthesis by intestinal cells. Furthermore, they indicate that p38 activity is required at a step distal to arachidonate release, most likely COX-2 upregulation, because exogenous arachidonate did not restore PGE(2) synthesis.

Arachidonic Acid↗
PubMed1995

c-myc mRNA is stabilized by deprivation of amino acids in primary cultured rat hepatocytes.

We investigated whether the expression of growth-related genes could be changed in primary cultured hepatocytes in response to changes in the nutritional environment. Hepatocytes were isolated from the liver of growing rats after collagenase perfusion and cultured in Williams' E medium (WE medium) containing 5% calf serum, 10(-7) M insulin and 10(-6) M dexamethasone for 24 h. When amino acids were removed from the culture, the level of c-myc mRNA increased more than 18-fold within 2-3 h, whereas replenishment of the amino acids to the medium caused rapid decrease in the mRNA level. We found that the half-life of the c-myc mRNA was prolonged more than 6-fold in the absence of amino acids. The mRNA levels of other proteins, such as ornithine decarboxylase, c-Ha-ras and actin, and their half-lives were not affected by amino acids. It is known that a short-lived protein is involved in the degradation of c-myc mRNA. In fact, the addition of cycloheximide to cultured hepatocytes increased the level of c-myc mRNA either in the presence or absence of amino acids, though the levels of other mRNAs were not changed significantly. These results suggest that the synthesis of the short-lived protein is suppressed and the c-myc mRNA is thereby stabilized in the absence of amino acids.

Actins↗
PubMed1997

Inhibition of tumour necrosis factor production in endotoxin-stimulated human mononuclear leukocytes by the prostacyclin analogue iloprost: cellular mechanisms.

The prostacyclin analogue iloprost has been shown to inhibit effectively TNF-alpha production in human peripheral blood mononuclear leukocytes (PBMC) stimulated with bacterial lipopolysaccharide (LPS). The current paper set out to analyse further the possible mechanisms involved in the regulation of TNF-alpha synthesis by iloprost. Healthy human PBMC were challenged with Escherichia coli LPS and assessed for TNF-alpha gene transcription, mRNA stability and protein secretion. Iloprost reduced both steady-state TNF-alpha mRNA expression and protein release as assessed by Northern blot analysis, polymerase chain reaction and enzyme immunoassay. This effect was related both to a reduction of TNF-alpha transcriptional activity (as evaluated by nuclear run-on transcription analysis) and a decrease in TNF-alpha mRNA stability (as assessed by serial Northern blot analysis of TNF-alpha mRNA content in PBMC blocked with actinomycin D). When collectively assessed, these data demonstrate that iloprost regulates TNF-alpha synthesis at both transcriptional and post-transcriptional level.

Cells, Cultured↗
PubMed1994

Induction of human interleukin-1 gene expression by retinoic acid and its regulation at processing of precursor transcripts.

Retinoic acid (RA), we show, induces in peripheral blood mononuclear cells a transient wave of newly transcribed, unstable interleukin-1 alpha (IL-1 alpha) and IL-1 beta mRNA. Tumor necrosis factor-alpha mRNA, by contrast, is expressed in multiple waves. IL-1 genes are primary targets for RA. Most IL-1 beta gene transcription induced by RA fails to yield mature mRNA. Instead, precursor transcripts accumulate, detected by ribonuclease protection analysis. The flow of precursors into IL-1 beta mRNA becomes inhibited during induction. When translation is blocked, e.g. by cycloheximide, expression of IL-1 beta mRNA is superinduced by 2 orders of magnitude. Superinduction is dependent on transcription, yet is unaccompanied by increased primary transcription or mRNA stability. Instead, processing of unstable IL-1 beta precursor transcripts into mature mRNA is greatly facilitated. Control is not narrowly localized within precursors: splicing of distinct exons and intron excision are enhanced by cycloheximide. Pre-mRNA processing thus is a limiting step in RA-induced IL-1 beta gene expression. This regulation is specific for RA: when induced by phorbol ester, IL-1 beta gene expression is also superinduced by cycloheximide but that response is accompanied by enhanced mRNA stability. Thus, IL-1 beta gene transcription is induced by RA, yet, unlike for other primary target genes, mRNA expression is regulated at pre-mRNA processing.

Cells, Cultured↗
PubMed1999

Epidermal growth factor regulates glucose metabolism through lactate dehydrogenase A messenger ribonucleic acid expression in cultured porcine Sertoli cells.

In a previous work, we reported that lactate dehydrogenase A4 (LDH A4) activity is a key step in the stimulatory effect of epidermal growth factor (EGF) on lactate production in cultured Sertoli cells. Here, we further investigated the regulatory mechanisms involved in EGF action on LDH A mRNA expression. Steady-state levels of LDH A mRNA analyzed by Northern blot hybridization were induced to 2. 9-fold in response to a 36-h incubation with EGF (ED(50) = 4 ng/ml, 0.63 x 10(-9) M). Whether EGF-induced increases of LDH A mRNA levels are the result of increased transcription and/or altered mRNA stability was investigated. The decay curves for the 1.5-kilobase LDH A mRNA transcript in Sertoli cells were not different in the absence or presence of EGF, suggesting that EGF did not affect LDH A mRNA stability. Inhibitors of protein synthesis (cycloheximide) and RNA synthesis (actinomycin D, and 5,6-dichloro-1-beta-ribofuranosyl benzimidazole) completely abrogated the EGF-induced LDH A mRNA expression, indicating that EGF increased LDH A mRNA levels through a transcriptional mechanism, which probably involves protein synthesis. Finally, the partial inhibitory effect of a protein kinase C (PKC) inhibitor, bisindolylmaleimide, on EGF-stimulated LDH A mRNA supports a partial involvement of PKC in the action of the growth factor. Since EGF is produced in Sertoli and in germ cells, its action is probably exerted in a context of a local control. As EGF also regulates other parameters involved in glucose metabolism, its effect on LDH A might be viewed in a general context related to the control of energy metabolism by the growth factor in the testicular cells.

Animals↗
PubMed1999

Premature termination codon in the aggrecan gene of nanomelia and its influence on mRNA transport and stability.

AIM: To analyze the influence of the premature termination codon on mRNA transport and stability METHODS: Chondrocyte mRNA was isolated from homozygous and heterozygous nanomelic 17-days old embryos and examined by RT-PCR analysis. To analyze aggrecan mRNA stability, mRNA synthesis was inhibited with DRB [5,6 dichloro-1-(-D-ribofuranosyl benzimidazole)], a specific inhibitor of RNA polymerase II. Visualization of the aggrecan alleles was performed by in situ hybridization. RESULTS: The level of mutant aggrecan mRNA within the nucleus was equal to that of the control, but no mutant mRNA was observed in the cytoplasm. RT-PCR revealed that the mutant transcript was only detectable in the nucleus, compared with house-keeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene or collagen type II. A restriction site induced by premature termination codon TAA allowed the distinction of normal and mutant transcripts in chondrocytes derived from embryos heterozygous for the nanomelic mutation. After the treatment with DRB, identical decay rates were demonstrated for both transcripts within the heterozygous nucleus. In situ hybridization showed no abnormal mRNA accumulation. CONCLUSION: This is the first evidence suggesting that the transcript of the mRNA with the premature termination codon within an exon does exit the nucleus.

Aggrecans↗
PubMed2001

Apoptosis is associated with modifications of bcl-2 mRNA AU-binding proteins.

The expression of genes requiring finely tuned control is regulated by a posttranscriptional mechanism involving mRNA A + U-rich elements (AREs) cooperating with ARE-binding proteins (AUBPs) in modulation of mRNA stability. We reported previously that an ARE in the bcl-2 mRNA 3'-untranslated region (3'-UTR) had destabilizing activity and was involved in bcl-2 downregulation during apoptosis in vitro. Here we demonstrate that the bcl-2 ARE complexes with a number of specific AUBPs, whose pattern undergoes changes following application of apoptotic stimuli. The caspase inhibitor Z-VAD-fmk strongly attenuates both bcl-2 mRNA decay and bcl-2 AUBP pattern changes elicited by apoptotic stimuli, indicating the involvement of bcl-2 AUBPs in bcl-2 mRNA stability control.

3' Untranslated Regions↗