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PubMed2002

A self-fulfilling prophecy: C-reactive protein attenuates nitric oxide production and inhibits angiogenesis.

BACKGROUND: Given the central importance of nitric oxide (NO) in the development and clinical course of cardiovascular diseases, we sought to determine whether the powerful predictive value of C-reactive protein (CRP) might be explained through an effect on NO production. METHODS AND RESULTS: Endothelial cells (ECs) were incubated with recombinant CRP (0 to 100 microg/mL, 24 hours), and NO and cyclic guanosine monophosphate (cGMP) production was assessed. The effects of CRP on endothelial NO synthase (eNOS) protein, mRNA expression, and mRNA stability were also examined. In a separate study, the effects of CRP (25 microg/mL) on EC cell survival, apoptosis, and in vitro angiogenesis were evaluated. Incubation of ECs with CRP resulted in a significant inhibition of basal and stimulated NO release, with concomitant reductions in cGMP production. CRP caused a marked downregulation of eNOS mRNA and protein expression. Actinomycin D studies suggested that eNOS downregulation was related to decreased mRNA stability. In conjunction with a decrease in NO production, CRP inhibited both basal and vascular endothelial growth factor-stimulated angiogenesis as assessed by EC migration and capillary-like tube formation. CRP did not induce EC survival but did, however, promote apoptosis in a NO-dependent fashion. CONCLUSIONS: CRP, at concentrations known to predict adverse vascular events, directly quenches the production of the NO, in part, through posttranscriptional effect on eNOS mRNA stability. Diminished NO bioactivity, in turn, inhibits angiogenesis, an important compensatory mechanism in chronic ischemia. Through decreasing NO synthesis, CRP may facilitate the development of diverse cardiovascular diseases. Risk reduction strategies designed to lower plasma CRP may be effective by improving NO bioavailability.

Apoptosis↗
PubMed2005

Gonadotropins upregulate the epidermal growth factor receptor through activation of mitogen-activated protein kinases and phosphatidyl-inositol-3-kinase in human ovarian surface epithelial cells.

Gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) have been implicated as probable risk factors in epithelial ovarian carcinomas, most of which are derived from ovarian surface epithelium (OSE). Since epidermal growth factor (EGF) increases the growth of ovarian surface epithelial cells, we determined the effect of gonadotropins on the expression of epidermal growth factor receptor (EGFR). We investigated the basal levels of EGFR mRNA and protein, and the mechanisms involved in the regulation of EGFR at the transcriptional and translational levels by FSH and LH. The immortalized OSE cell lines (IOSE) derived from normal OSE cells by transfecting SV40 T-antigen (IOSE-80 and IOSE-80PC, a post-crisis line) and ovarian cancer cell lines were employed. A significantly lower level of EGFR was observed in both IOSE-80 and IOSE-80PC cells when compared with the ovarian cancer cell lines, OVCAR-3 and SKOV-3. Treatment of IOSE-80PC cells with FSH and LH (10(-7) and 10(-6) g/ml) resulted in a significant increase in EGFR mRNA at 24 h and EGFR protein at 48 h, whereas the treatment with gonadotropins for 24-48 h induced a mild increase in EGFR in OVCAR-3, but not in SKOV-3 cells. In addition, IOSE-80PC cells treated with gonadotropins and EGF (10 nM) exhibited an additive stimulation of mitogenesis. Further, FSH and LH significantly increased activities of various kinases at 5-10 min, and pre-treatments with LY294002 (an inhibitor of PI3K) or PD98059 (an inhibitor of ERK1/2) partially blocked the gonadotropin-induced up-regulation of EGFR in IOSE-80PC cells. We investigated whether the effect of gonadotropins on EGFR mRNA levels is induced by increased transcription and/or by altered mRNA stability. Treatment of IOSE-80PC cells with FSH (10(-7) and 10(-6) g/ml) significantly enhanced the activity of the EGFR promoter (120 and 140% increase, respectively) at 24 h, and treatment with LH (10(-7) g/ml) for 24 h induced an increase in the activity of EGFR promoter (30%) in these cells. On the other hand, LH resulted in a significant increase in EGFR mRNA stability in the decay curves. Taken together, these results suggest that the effect of gonadotropins on the expression of EGFR may affect cell growth via ERK-1/-2 and PI3K pathways in pre-neoplastic ovarian surface epithelial cells, and that FSH and LH increase EGFR mRNA by different mechanisms. The former increased EGFR gene transcription essentially, whereas the latter mainly enhanced EGFR mRNA stability.

Epithelial Cells↗
PubMed1993

The glucagon-insulin antagonism in the regulation of cytosolic protein binding to the 3' end of phosphoenolpyruvate carboxykinase mRNA in cultured rat hepatocytes. Possible involvement in the stabilization of the mRNA.

Since protein binding to the 3' end of mRNA is believed to be involved in the control of mRNA stability, the time course of alterations in glucagon-induced phosphoenolpyruvate-carboxykinase-mRNA (PCK) levels, in the absence and presence of insulin, was correlated with the time course of changes in the binding of cytosolic protein from 24-h cultured rat hepatocytes to the 3' end of PCK mRNA. PCK-mRNA levels were monitored by Northern blot analysis and protein binding was analyzed by an electrophoretic mobility-shift assay. In 24-h cultured rat hepatocytes, binding of cytosolic protein to the PCK-mRNA 3' end and PCK-mRNA levels were increased to a transient maximum at 2 h and 2-4 h, respectively, by a 1-nM glucagon treatment, added with a change of medium. 100 nM insulin, added simultaneously with glucagon, reduced the glucagon-induced maximum of protein binding by 80% and the increase of PCK mRNA by about 30%. In controls without hormonal treatment protein binding at 1 h was also increased; this increase was prevented by insulin. 100 nM insulin, added 1 h after glucagon, reversed protein binding to the 3' end of PCK mRNA to nearly initial levels within 1 h and impaired the glucagon-induced increase in PCK-mRNA levels by 30%. The transcriptional inhibitor cordycepin, added 1 h after glucagon, did not prevent the further increase in glucagon-enhanced protein binding nor its reversal by insulin. It did, however, prevent a further significant increase in PCK mRNA. Hormonally regulated protein binding could be localized to the 256 distal bases of the PCK-mRNA 3' end. The proximal 466 bases of the PCK-mRNA 3' end as well as the 1050 bases of the histone-H1(0)-mRNA 3' end and the 1200 bases of the arylsulfatase-A-mRNA 3' end also bound cytosolic protein(s), but this protein binding was not altered by treatment with glucagon or insulin. The 3' end of PCK, arylsulfatase A and H1(0) mRNA exhibited strong binding of cytosolic protein(s) from diverse rat tissues such as heart, liver and lung as well as Fao rat hepatoma cells. Cytosolic protein(s) from spleen showed weak binding and proteins from HeLa and U937 tumor cells did not bind. Protein binding was most prominent with the 3' end of PCK mRNA and cytosolic extracts from liver.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2006

AU-rich elements and associated factors: are there unifying principles?

The control of mRNA stability is an important process that allows cells to not only limit, but also rapidly adjust, the expression of regulatory factors whose over expression may be detrimental to the host organism. Sequence elements rich in A and U nucleotides or AU-rich elements (AREs) have been known for many years to target mRNAs for rapid degradation. In this survey, after briefly summarizing the data on the sequence characteristics of AREs, we present an analysis of the known ARE-binding proteins (ARE-BP) with respect to their mRNA targets and the consequences of their binding to the mRNA. In this analysis, both the changes in mRNA stability and the lesser studied effects on translation are considered. This analysis highlights the multitude of mRNAs bound by one ARE-BP and conversely the large number of ARE-BP that associate with any particular ARE-containing mRNA. This situation is discussed with respect to functional redundancies or antagonisms. The potential relationship between mRNA stability and translation is also discussed. Finally, we present several hypotheses that could unify the published data and suggest avenues for future research.

3' Untranslated Regions↗
PubMed1993

Stabilization of a protein-tyrosine phosphatase mRNA upon mitogenic stimulation of T-lymphocytes.

The expression of a non-receptor type protein-tyrosine phosphatase (the T-cell phosphatase or PTP-S) which shows homology with basic domains of Fos and Jun, was investigated upon mitogenic stimulation of rat splenic T lymphocytes. As studied by Northern blot analysis of total cellular RNA, mitogenic stimulation of T lymphocytes by concanavalin A resulted in an increase in the level of PTP-S mRNA; there was little or no change in the level of mRNA coding for PTP-1 (which is also a non-receptor type tyrosine phosphatase). Maximum increase of about 3-fold in the level of PTP-S mRNA occurred after 72 h of mitogenic stimulation. Mitogenic stimulation did not increase the level of PTP-S transcripts in the nucleus. The half-life of PTP-S mRNA in unstimulated lymphocytes was about 25 min which increased to 5 h after mitogenic stimulation. An inhibitor of protein synthesis, cycloheximide, increased the level of PTP-S transcripts by 6-fold in control lymphocytes but did not increase the level of PTP-1 transcripts. Treatment with cycloheximide increased the half-life of PTP-S transcripts in resting lymphocytes. The PTP-S gene product was identified as a 42 kDa polypeptide by immunoblotting. The level of PTP-S gene product increased upon mitogenic stimulation of lymphocytes by Con A and reached a maximum after 72 h, as determined by immunoblotting. These results suggest that post-transcriptional regulation of mRNA stability is an important factor in controlling the level of this phosphatase mRNA during mitogenic stimulation of T-lymphocytes.

Animals↗
PubMed2000

Stress regulation of adrenocorticosteroid receptor gene transcription and mRNA expression in rat hippocampus: time-course analysis.

Neuronal glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) proteins mediate the transcriptional effects of circulating glucocorticoids. These receptors bind the same DNA response element, yet mediate quite different cellular functions. The present study tests the hypothesis that acute and chronic stress, which cause increases in glucocorticoids sufficient to bind the GR, will regulate expression of the GR and MR genes in the hippocampal formation. Analysis of MR gene transcription using an intronic MR probe revealed a transient 50% decrease in MR hnRNA in CA1, CA3 and dentate gyrus at 60-120 min post-stress, consistent with glucocorticoid down-regulation of the MR gene. However, no changes were seen in full-length MR mRNA at any post-stress time point. In contrast, GR hnRNA was not affected by acute stress, but GR mRNA was decreased 120 min post stress in all hippocampal subregions. Chronic stress exposure down-regulated GR mRNA in CA3 only; effects were first evident 7 days post stress and persisted for the entire stress time-course (28 days). There was no evidence for down-regulation of GR hnRNA or MR hnRNA/mRNA at any point in the chronic stress regimen. The transient decrease in MR hnRNA in the absence of mRNA changes suggests increased MR mRNA stability. In contrast, acute stress decreases the availability of GR mRNA without demonstrably affecting transcription, suggesting reduced GR mRNA stability. The results suggest that acute stress alters GR mRNA expression by largely post-transcriptional mechanisms. However, elevations in basal corticosterone secretion seen following chronic stress are not sufficient to markedly down-regulate GR/MR expression in a long-term fashion.

Acute Disease↗
PubMed2025

Iron-mediated post-transcriptional regulation in Toxoplasma gondii.

Iron is required to support almost all life; however, levels must be carefully regulated to maintain homeostasis. Although the obligate parasite Toxoplasma gondii requires iron, how it responds upon iron limitation has not been investigated. Here, we show that iron depletion triggers significant transcriptional changes in the parasite, including in iron-dependent pathways. We find that a subset of T. gondii transcripts contain stem-loop structures, which have been associated with post-transcriptional iron-mediated regulation in other cellular systems. We validate one of these (found in the 3' UTR of TGME49_261720) using a reporter cell line. We show that the presence of the stem-loop-containing UTR is sufficient to confer accumulation at the transcript and protein levels under low iron. This response is dose and time-dependent and is specific for iron. The accumulation of transcript is likely driven by an increased reporter mRNA stability under low iron. Interestingly, we find iron-mediated changes in mRNA stability in around 400 genes. To examine the potential mechanism of this stability, we tested aconitase interaction with mRNA in low iron and found 43 enriched transcripts, but no specific interaction with our reporter UTR. However, the endogenous UTR led to maintenance of protein levels and increased survival of the parasite under low iron. Our data demonstrate the existence of iron-mediated post-transcriptional regulation in Toxoplasma for the first time; and suggests iron-mediated regulation may be important to the parasite in low iron environments.

Toxoplasma↗
PubMed1992

Post-transcriptional regulation of collagenase and stromelysin gene expression by epidermal growth factor and dexamethasone in cultured human fibroblasts.

Epidermal growth factor (EGF) is a ubiquitous fibroblast mitogen which also stimulates the synthesis of the extracellular matrix degrading metalloproteinases, collagenase, and stromelysin. Using primary cultures of human skin fibroblast, we show that these metalloproteinase mRNAs are coordinately up-regulated by EGF; and that dexamethasone, a potent inhibitor of collagenase and stromelysin synthesis, coordinately down-regulates these EGF-induced mRNAs. Nuclear run-on assays showed that EGF increased transcription of collagenase and stromelysin approximately 2-fold over the untreated control, while repression by dexamethasone was difficult to detect. However, steady state mRNA levels were induced approximately 10-fold by EGF and co-treatment with dexamethasone decreased them to below control levels, suggesting modulation of mRNA stability. Thus, we measured the half-life of these mRNAs using "pulse-chase" methodology. Typically, the half-life of EGF-induced collagenase and stromelysin mRNAs was approximately 30 h, and co-treatment with dexamethasone decreased the half-life of these mRNAs by 30-50%. Additionally, we found that the transcription inhibitor DRB stabilized EGF-induced metalloproteinase mRNAs, suggesting an mRNA degradation pathway which requires transcription. Thus our data demonstrate that collagenase and stromelysin are coordinately regulated by EGF and by dexamethasone, primarily at the level of metalloproteinase mRNA stability.

Blotting, Northern↗
PubMed2000

Molecular characterization of the SerL paralogs of Tetrahymena thermophila.

In the pond ciliate Tetrahymena thermophila, expression of genes encoding variant forms of the cell surface immobilization antigen (i-ag) is regulated by environmental conditions. Multiple isoforms of the L i-ags are found on the surface of cells grown at <20 degrees C as well as on the surface of rseC mutants which express SerL genes constitutively. Five cDNAs encoding variant L i-ags of rseC were sequenced and their expression studied. Two additional SerL genes from natural isolates were sequenced. Members of the SerL family encode polypeptides with 148, 316, or 371 amino acids, and the i-ags have two, five, or six imperfect repeats, respectively, flanked by putative ER translocation and GPI addition signals. Each repeat contains six periodic cysteines, in contrast to eight or ten in other i-ags of T. thermophila. At least three of the five genes constitutively expressed in rseC mutants are differentially expressed in cells expressing other i-ags. Northern analysis and RT-PCR indicate that expression of some members of the SerL family is regulated by both transcription and mRNA stability while another member is regulated primarily by mRNA stability.

Amino Acid Sequence↗
PubMed1988

Oxytocin and vasopressin gene expression during gestation and lactation.

Levels of rat hypothalamic oxytocin (OT) and vasopressin (AVP) mRNA were determined during gestation and lactation using a densitometric hybridization assay. Pregnancy induced a gradual rise in OT mRNA, reaching, by day 18, a level exceeding control by a factor of 2.5. Throughout the lactation period OT mRNA remained elevated at levels corresponding to 3 times that of control. Surprisingly, the dynamics of AVP mRNA accumulation paralleled closely the profile observed for OT mRNA throughout the time period studied. We conclude that in late pregnancy and lactation the expression of both, the OT and the AVP gene is stimulated in parallel by mechanisms operating at a pretranslational level, involving increased gene transcription or mRNA stabilization, or both. Further characterization of the two mRNA species revealed that both mRNAs are endowed with very long poly(A) tails of greater than 200 nucleotides, under conditions of both, normal and stimulated states of gene expression. The role, if any, of the prolonged poly(A) tails in mRNA stability remains to be determined.

Animals↗
PubMed1989

Altered binding of human histone gene transcription factors during the shutdown of proliferation and onset of differentiation in HL-60 cells.

Two sites of protein-DNA interaction have been identified in vivo and in vitro in the proximal promoter regions of an H4 and an H3 human histone gene. In proliferating cells, these genes are transcribed throughout the cell cycle, and both the more distal site I and the proximal site II are occupied by promoter-binding factors. In this report we demonstrate that during the shutdown of proliferation and onset of differentiation of the human promyelocytic leukemia cell line HL-60 into cells that exhibit phenotypic properties of monocytes, histone gene expression is down-regulated at the level of transcription. In vivo occupancy of site I by promoter factors persists in the differentiated HL-60 cells, but protein-DNA interactions at site II are selectively lost. Furthermore, in vitro binding activity of the site II promoter factor HiNF-D is lost in differentiated cells, and nuclear extracts from differentiated cells do not support in vitro transcription of these histone genes. Our results suggest that the interaction of HiNF-D with proximal promoter site II sequences plays a primary role in rendering cell growth-regulated histone genes transcribable in proliferating cells. It appears that while cell-cycle control of histone gene expression is mediated by both transcription and mRNA stability, with the shutdown of proliferation and onset mRNA stability, with the shutdown of proliferation and onset of differentiation, histone gene expression is regulated at the transcriptional level.

Base Sequence↗
PubMed1998

The role of calcium in the transcriptional and posttranscriptional regulation of the gonadotropin-releasing hormone gene in GT1-7 cells.

The role of calcium in the regulation of GnRH gene expression and the mechanism for its effects were examined in the present study. Using the immortalized hypothalamic GT1-7 cell line, which synthesizes and secretes GnRH, we demonstrated by ribonuclease protection assay and Northern blot analysis that these cells respond to treatment with the calcium ionophores ionomycin and A23187 with an inhibition of transcription of the GnRH gene and decreases in GnRH messenger RNA (mRNA) levels. Ionomycin treatment caused the GnRH mRNA half-life to decrease from 25 to 9 h, concomitant with a decrease in mRNA poly(A) tail length, suggesting that ionomycin causes a decrease in GnRH mRNA stability. The ionomycin inhibitory effect on GnRH cytoplasmic mRNA levels was significantly inhibited in the presence of cycloheximide or the RNA synthesis inhibitor 5,6-dichloro-1beta-ribofuranosylbenzimidazole, indicating that novel protein/RNA synthesis is obligatory for this effect. We conclude that an increase in calcium levels caused by ionomycin inhibits GnRH gene expression at multiple levels, including GnRH gene transcription and mRNA stability in GT1-7 cells.

Animals↗
PubMed1981

The Role of the poly(A) sequence in mammalian messenger RNA.

The poly(A) sequence is added to 3' termini of nuclear RNA segments destined to become part of the mRNA, and may play an essential role in the selection of these segments. It appears to be required for at least some of the splicing events involved in mRNA processing. In the cytoplasm, the poly(A) segment is the target of a degradation process which causes its gradual shortening, and leads to a heterogeneous steady-state poly(A)-size distribution. Complete loss of the poly(A) is probably followed by inactivation of the mRNA, since chains depleted of poly(A) do not accumulate in the cells. A role for this sequence in the promotion of mRNA stability is suggested by the behavior of globin mRNA depleted of poly(A) after injection into frog oocytes. The poly(A) shortening process may be part of the mRNA inactivation mechanism, as indicated by the greater sensitivity to degradation of the poly(A) of some short-lived mRNAs. However, the stochastic mRNA decay implies that new and old mRNA chains, with long and short poly(A) segments, respectively are equally susceptible to inactivation. The poly(A)-lacking histone mRNAs are stable only in cells engaged in DNA replication. Present knowledge favors a role for poly(A) in the control of mRNA stability. Loss of this sequence could be controlled through modulation of poly(A)-protein interactions or through masking of a sequence directly adjacent to the poly(A). In the nucleus, the poly(A) sequence could also serve as stabilizing agent, but, in addition, it might interact with the splicing machinery.

Actins↗
PubMed2005

The RNA binding protein TIAR is involved in the regulation of human iNOS expression.

Human inducible NO synthase (iNOS) expression is regulated by post-transcriptional mechanisms. The 3'-untranslated region (3'-UTR) of the human iNOS mRNA contains AU-rich elements (ARE), which are known to be important for the regulation of mRNA stability. The 3'-UTR of the human iNOS mRNA has been shown to regulate human iNOS mRNA expression post-transcriptionally. One RNA-binding protein known to interact with AREs and to regulate mRNA stability is the T cell intracellular antigen-1-related protein (TIAR). In RNA binding studies TIAR displayed high affinity binding to the human iNOS 3'-UTR sequence. In RNase protection experiments, the cytokine incubation needed for iNOS expression did not change TIAR expression in DLD-1 cells. However, overexpression of TIAR in human DLD-1 colon carcinoma cells resulted in enhanced cytokine-induced iNOS expression. In conclusion, TIAR seems to be involved in the post-transcriptional regulation of human iNOS expression.

3' Untranslated Regions↗
PubMed2003

Relationship of trichothecene structure to COX-2 induction in the macrophage: selective action of type B (8-keto) trichothecenes.

The trichothecene mycotoxin deoxynivalenol (DON, vomitoxin), when at partially cytotoxic concentrations, induces cyclooxygenase-2 (COX-2) expression by promoting transcriptional activity and mRNA stability via mitogen-activated protein kinase (MAPK) signaling pathways. The purpose of this study was to test the hypothesis that trichothecenes differentially affect COX-2 gene expression and that these effects were related to MAPK activation. Representative members of the three major trichothecene families (A, B, and D) were compared for their capacity to induce COX-2 in the RAW 264.7 murine macrophage cell line. When cells were treated with concentrations that inhibited the 3-(4,5-di-methylthizol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) viability response by 20% (IC20), Type B trichothecenes including DON, 15-acetyl-DON, 3-acetyl-DON, and fusarenon-X were found to be effective inducers of COX-2 mRNA expression, whereas equitoxic Type A and Type D trichothecenes had markedly less effects. To compare effects of COX-2 gene transactivation and mRNA stabilization, luciferase reporter vectors containing 5'-promoter or 3'-untranslated regions of the gene, respectively, were transfected into RAW 264.7 cells and the effects of various trichothecenes on luciferase activities were measured. Type B but not Type A or D toxins at concentrations up to the MTT IC50 enhanced luciferase activities, indicating preferential COX-2 transcriptional activation and mRNA stabilization by this trichothecene subset. At their respective IC20s, Type B trichothecenes also significantly activated the three major MAPK families, whereas Type A and D did not. Blocking ERK and p38 with chemical inhibitors significantly suppressed Type B-induced COX-2 expression. Although JNK reportedly contributes to COX-2 expression in the other signaling models, transfection with the dominant negative JNK vector did not diminish the COX-2 expression. Taken together, Type B trichothecenes selectively enhanced transcription and stabilization of the COX-2 gene, and this was mediated by the ERK 1/2 and p38 signaling pathways. Selective action on COX-2 might contribute to unique pathologic manifestations associated with Type B trichothecene-mediated immunotoxicity.

Animals↗
PubMed2001

Y box-binding factor promotes eosinophil survival by stabilizing granulocyte-macrophage colony-stimulating factor mRNA.

Short-lived peripheral blood eosinophils are recruited to the lungs of asthmatics after allergen challenge, where they become long-lived effector cells central to disease pathophysiology. GM-CSF is an important cytokine which promotes eosinophil differentiation, function, and survival after transit into the lung. In human eosinophils, GM-CSF production is controlled by regulated mRNA stability mediated by the 3' untranslated region, AU-rich elements (ARE). We identified human Y box-binding factor 1 (YB-1) as a GM-CSF mRNA ARE-specific binding protein that is capable of enhancing GM-CSF-dependent survival of eosinophils. Using a transfection system that mimics GM-CSF metabolism in eosinophils, we have shown that transduced YB-1 stabilized GM-CSF mRNA in an ARE-dependent mechanism, causing increased GM-CSF production and enhanced in vitro survival. RNA EMSAs indicate that YB-1 interacts with the GM-CSF mRNA through its 3' untranslated region ARE. In addition, endogenous GM-CSF mRNA coimmunoprecipitates with endogenous YB-1 protein in activated eosinophils but not resting cells. Thus, we propose a model whereby activation of eosinophils leads to YB-1 binding to and stabilization of GM-CSF mRNA, ultimately resulting in GM-CSF release and prolonged eosinophil survival.

3' Untranslated Regions↗
PubMed1993

Posttranscriptional regulation of GAP-43 gene expression in PC12 cells through protein kinase C-dependent stabilization of the mRNA.

We have previously shown that nerve growth factor (NGF) selectively stabilizes the GAP-43 mRNA in PC12 cells. To study the cellular mechanisms for this post-transcriptional control and to determine the contribution of mRNA stability to GAP-43 gene expression, we examined the effects of several agents that affect PC12 cell differentiation on the level of induction and rate of degradation of the GAP-43 mRNA. The NGF-mediated increase in GAP-43 mRNA levels and neurite outgrowth was mimicked by the phorbol ester TPA, but not by dibutyryl cAMP or the calcium ionophore A12783. Downregulation of protein kinase C (PKC) by high doses of phorbol esters or selective PKC inhibitors prevented the induction of this mRNA by NGF, suggesting that NGF and TPA act through a common PKC-dependent pathway. In mRNA decay studies, phorbol esters caused a selective 6-fold increase in the half-life of the GAP-43 mRNA, which accounts for most of the induction of this mRNA by TPA. The phorbol ester-induced stabilization of GAP-43 mRNA was blocked by the protein kinase inhibitor polymyxin B and was partially inhibited by dexamethasone, an agent that blocks GAP-43 expression and neuronal differentiation in PC12 cells. In contrast, the rates of degradation and the levels of the GAP-43 mRNA in control and TPA-treated cells were not affected by cycloheximide treatment. Thus, changes in GAP-43 mRNA turnover do not appear to require continuous protein synthesis. In conclusion, these data suggest that PKC activity regulates the levels of the GAP-43 mRNA in PC12 cells through a novel, translation-independent mRNA stabilization mechanism.

Animals↗
PubMed1994

mRNAs can be stabilized by DEAD-box proteins.

Eubacterial messenger RNAs are synthesized and translated simultaneously; moreover the speed of ribosomes usually matches that of RNA polymerase. We report here that when in Escherichia coli the host RNA polymerase is replaced by the eightfold faster bacteriophage T7 enzyme for the transcription of the lacZ gene, the beta-galactosidase yield per transcript is depressed 100-fold. But the overexpression of DEAD-box proteins greatly improves this low yield by stabilizing the corresponding transcripts. More generally, it stabilizes inefficiently translated E. coli mRNAs. Ribosome-free mRNA regions, such as those lying behind the fast T7 enzyme or between successive ribosomes on inefficiently translated transcripts, are often unstable and we propose that DEAD-box proteins protect them from endonucleases. These results pinpoint the importance of transcription-translation synchronization for mRNA stability, and reveal an undocumented property of DEAD-box RNA helicases. These proteins have been implicated in a variety of processes involving RNA but not mRNA stability.

Amino Acid Sequence↗