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PubMed1996

Role of highly conserved pyrimidine-rich sequences in the 3' untranslated region of the GAP-43 mRNA in mRNA stability and RNA-protein interactions.

We have shown previously that the mRNA for the growth-associated protein GAP-43 is selectively stabilized during neuronal differentiation. In this study, we explored the role of its highly conserved 3' untranslated region (3'UTR) in mRNA stability and RNA-protein interactions. The 3'UTRs of the rat and chicken GAP-43 mRNAs show 78% sequence identity, which is equivalent to the conservation of their coding regions. In rat PC12 cells stably transfected with the full-length rat or chicken GAP-43 cDNAs, the transgene mRNAs decayed with same half-life of about 3 h. The GAP-43 3'UTR also caused the rabbit beta-globin mRNA to decay with a half-life of 4 h, indicating that the major determinants for GAP-43 mRNA stability are localized in its highly conserved 3'UTR. Three brain cytosolic RNA-binding proteins (molecular mass 40, 65 and 95 kDa) were found to interact with both the rat and chicken GAP-43 mRNAs. These RNA-protein interactions were specific and involved pyrimidine-rich sequences in the 3'UTR. Like the GAP-43 mRNA, the activity of these proteins was enriched in brain and increased during development. We propose that highly conserved pyrimidine-rich sequences in the 3'UTR of this mRNA regulate GAP-43 gene expression via interactions with specific RNA-binding proteins.

Animals↗
PubMed1995

IFN-gamma priming of monocytes enhances LPS-induced TNF production by augmenting both transcription and MRNA stability.

The induction of cytokine expression in monocytes/macrophages by bacterial endotoxin or lipopolysaccharide is a critical, highly regulated host defence response. The augmentation of LPS responses by interferon gamma (IFN-gamma), referred to as priming, is well established. However, the mechanism(s) by which priming occurs is poorly defined. Using tumour necrosis factor (TNF) induction as a model, experiments were designed to analyse in detail the priming effect on the LPS response in human monocytes. Priming by IFN-gamma was primarily manifested at the level of TNF mRNA accumulation. IFN-gamma pre-treatment affected the magnitude rather than the sensitivity of the LPS response. Priming occurred after several hours of treatment, and the primed state was induced by either IFN-gamma or GM-CSF, but not M-CSF. Primed monocytes transcribed TNF mRNA at a higher rate than freshly isolated monocytes upon activation with LPS. The increased transcriptional rate correlated with a marked increase in nuclear factor-kappa B activity in these cells as determined by electrophoretic mobility shift assay using a consensus NF-kappa B oligonucleotide. An additional significant finding was than TNF mRNA induced in primed cells was much more stable than in unprimed cells (T1/2 increased 6-8-fold). Consistent with the increased mRNA stability, the duration of mRNA accumulation was longer following LPS stimulation in primed monocytes, in addition to being of greater magnitude. Finally, primed and unprimed cells possessed a differential sensitivity to the kinase inhibitor H-89. H-89 substantially suppressed LPS-induced TNF mRNA accumulation in unprimed cells, but had no effect on primed monocytes following LPS stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed2001

K-Ras-mediated increase in cyclooxygenase 2 mRNA stability involves activation of the protein kinase B1.

Cyclooxygenase (COX) 2 expression is regulated via the Ras signaling pathway, and induction of mutated Ras rapidly increases COX-2 levels in intestinal epithelial cells. Protein kinase B (Akt/PKB) is an important effector of Ras signaling and a critical component of Ras-mediated transformation. Here we investigate the role of Akt/PKB in K-Ras-mediated induction of COX-2. Rat intestinal epithelial cells (IEC-6) were transfected with an inducible K-RasVal12 cDNA (IEC-iK-Ras cells). Addition of 5 mM isopropyl-1-thio-beta-D-galactopyranoside induced the expression of K-RasVal12, followed by increased activity of extracellular signal-regulated kinase and Akt/PKB. COX-2 levels were dramatically increased after induction of K-RasVal12. Inhibition of MAPK/ERK kinase activity by PD 98059 completely blocked the K-Ras-mediated induction of COX-2, whereas inhibition of PI3K/Akt/PKB activity with LY 294002 or by expressing a dominant negative Akt (Akt-K179M) partially blocked the induction of COX-2 by K-Ras. Transient transfection of cells with phosphatidylinositol 3-kinase and Akt expression vectors revealed that PI3/Akt/PKB activity predominantly regulates the stability of COX-2 mRNA. Thus, Akt/PKB activity is involved in K-Ras-induced expression of COX-2 and stabilization of COX-2 mRNA largely depends on the activation of Akt/PKB.

Animals↗
PubMed2004

Down-regulation of soluble guanylyl cyclase expression by cyclic AMP is mediated by mRNA-stabilizing protein HuR.

We analyzed whether the cyclic AMP induced down-regulation of the nitric oxide (NO) receptor soluble guanylyl cyclase (sGC) is mediated by the mRNA-protecting protein HuR. Exposure (up to 24 h) of isolated rat aortic segments to the activator of adenylyl cyclase, forskolin (10 microM), and to both activators of cAMP-stimulated protein kinase (PKA), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphorothioate, Spisomer (Sp-5,6-DCl-cBIMPS; 400 nM), and N6-phenyl-cAMP (10 microM), strongly reduced sGCalpha1beta1 and HuR protein and mRNA expression in a time-dependent and actinomycin D (10 microM)-sensitive fashion. In vitro degradation of sGCalpha1 and beta1 poly(A)+ mRNA by native rat aortic protein was markedly increased by pretreatment of intact aortas with forskolin. Native protein extract from rat aorta shifted the electrophoretic mobility of biotin-labeled riboprobes from the 3'-untranslated region of sGCalpha1 and beta1 mRNA, and these bands was supershifted by a monoclonal antibody directed against the mRNA-stabilizing protein HuR. Forskolin decreased the HuR-sGCalpha1 and beta1 mRNA interaction and HuR protein expression in rat aorta, and this was prevented by the PKA inhibitory cAMP analog 3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In cultured smooth muscle cells from rat aorta, forskolin induced a rapid increase in Fos/p-Fos protein levels and activator protein 1 (AP-1) binding activity. Inhibition of this transcription factor by an AP-1 decoy prevented the forskolin-induced down-regulation of HuR. We conclude that forskolin/cAMP decrease the expression of heterodimeric sGC in rat aortic smooth muscle cells via activation of Fos/AP-1, which decreases the expression of HuR and thus destabilizes the sGCalpha1 and beta1 mRNA.

3' Untranslated Regions↗
PubMed2005

Regulation of urokinase receptor mRNA stability by hnRNP C in lung epithelial cells.

Increased urokinase receptor (uPAR) expression as well as stabilisation of uPAR mRNA contribute to the pathogenesis of lung inflammation and neoplasia. Post-transcriptional regulation of uPAR mRNA involves interaction of both coding and 3'-UTR sequences with regulatory uPAR mRNA binding proteins (Bps). In order to identify novel regulatory interactions, we performed gel mobility shift and UV cross-linking assays and found two distinct uPAR mRNA-protein complexes. We identified a rapidly migrating 40 kDa uPAR mRNABp that selectively bound a 110 nucleotide (nt) fragment of the uPAR mRNA 3'UTR. Chimeric beta-globin/uPAR mRNA containing the 110 nt 40 kDa protein binding fragment destabilised stable beta-globin mRNA with a rate of decay identical to that of chimeric beta-globin/uPAR containing the full uPAR 3'UTR. The 40 kDa uPAR 3'UTR Bp was purified using poly (U) sepharose and identified as heterogeneous nuclear ribonucleoprotein C (hnRNPC). Finally, we confirmed its interaction with the uPAR mRNA 3' UTR by gel mobility supershift assay using an anti-hnRNPC antibody. Direct in vivo interaction of hnRNPC with the uPAR mRNA 3'UTR was demonstrated by immunoprecipitation and combined RT PCR-Southern blotting assay. Co-transfection of hnRNPC cDNA in Beas2B cells reversed destabilisation of chimeric beta-globin/uPAR 3'UTR mRNA and its over-expression also induced uPAR protein and mRNA expression through stabilisation of uPAR mRNA. These observations indicate a novel mechanism of uPAR gene regulation in lung epithelial cells in which cis elements within a 110 nt uPAR mRNA 3'UTR sequence interact with hnRNPC to regulate uPAR mRNA stability.

3' Untranslated Regions↗
PubMed2002

Modulation of eukaryotic mRNA stability via the cap-binding translation complex eIF4F.

Decapping by Dcp1 in Saccharomyces cerevisiae is a key step in mRNA degradation. However, the cap also binds the eukaryotic initiation factor (eIF) complex 4F and its associated proteins. Characterisation of the relationship between decapping and interactions involving eIF4F is an essential step towards understanding polysome disassembly and mRNA decay. Three types of observation suggest how changes in the functional status of eIF4F modulate mRNA stability in vivo. First, partial disruption of the interaction between eIF4E and eIF4G, caused by mutations in eIF4E or the presence of the yeast 4E-binding protein p20, stabilised mRNAs. The interactions of eIF4G and p20 with eIF4E may therefore act to modulate the decapping process. Since we also show that the in vitro decapping rate is not directly affected by the nature of the body of the mRNA, this suggests that changes in eIF4F structure could play a role in triggering decapping during mRNA decay. Second, these effects were seen in the absence of extreme changes in global translation rates in the cell, and are therefore relevant to normal mRNA turnover. Third, a truncated form of eIF4E (Delta196) had a reduced capacity to inhibit Dcp1-mediated decapping in vitro, yet did not change cellular mRNA half-lives. Thus, the accessibility of the cap to Dcp1 in vivo is not simply controlled by competition with eIF4E, but is subject to switching between molecular states with different levels of access.

Blotting, Western↗
PubMed1995

Regulation of interferon-gamma mRNA in a cytolytic T cell clone: Ca(2+)-induced transcription followed by mRNA stabilization through activation of protein kinase C or increase in cAMP.

Activation pathways inducing the expression of the interferon (IFN)-gamma gene in a cytotoxic T lymphocyte (CTL) clone were studied for their effects on transcription and on mRNA stability. IFN-gamma was secreted by the CTL clone in response to the Ca2+ ionophore ionomycin when used in conjunction with either protein kinase C (PKC)-activating phorbol 12-myristate 13-acetate (PMA) or with agents increasing cAMP, including prostaglandin E2. We describe that ionomycin induced IFN-gamma gene transcription, which was totally inhibited in the presence of cyclosporin A (CSA), an immunosuppressant forming a calcineurin-inhibiting complex with cyclophilin. Ionomycin did not, however, permit accumulation of IFN-gamma mRNA. Activation of PKC by PMA or of cAMP-dependent protein kinase through increase in cAMP had no transcription-inducing effect, either alone or in conjunction with ionomycin, as measured in run on assays of the IFN-gamma gene. When transcription of the IFN-gamma gene, initiated in the presence of ionomycin and an agent increasing intracellular cAMP, was inhibited by CSA in the absence of PKC or cAMP-dependent protein kinase activation, the IFN-gamma mRNA was rapidly degraded (half-life = 30 min). When either PKC was activated or intracellular cAMP was increased at the time of inhibition with CSA, a stabilizing effect was observed on IFN-gamma mRNA, which led to an increase in secreted IFN-gamma. These effects were selective, they did not affect the rate of transcription of the actin gene, nor the accumulation of actin mRNA. These results show that (i) post-transcriptional events can be critical for IFN-gamma expression in activated lymphocytes, and (ii) specific stabilization of IFN-gamma mRNA can be mediated by activation of two different protein kinases involved in T cell activation.

Animals↗
PubMed1989

Translation affects immunoglobulin mRNA stability.

When termination codons were introduced into exons of the gene for Ig mu chain, steady-state levels of mu mRNA were reduced, both at the pre-B cell stage and at the plasma cell stage. A termination codon in the variable region gene segment and a termination codon in the second exon of the constant region gene segment had effects of similar magnitude. When the termination codon was deleted, the original level of mRNA was restored. The rate of mu gene transcription was the same whether or not a termination codon was present. Therefore, the termination codons must reduce the amount of the mRNA by reducing its stability. Since the introduced termination codons prematurely terminate translation and, in so doing, change the ribosome load on the mRNA, we conclude that mu mRNA stability is conferred in part by ribosomal protection from enzymatic degradation. We propose that the differences in mu mRNA stability during B lymphocyte differentiation are due to different amounts of ribosomes available for translation.

B-Lymphocytes↗
PubMed2003

Beta-catenin inversely regulates vascular endothelial growth factor-D mRNA stability.

The angiogenic and lymphangiogenic vascular endothelial growth factor (VEGF)-D is the only member of the VEGF family that is not induced by hypoxia or by serum factors, but its induction is mediated by direct cell-cell contact. Here we show that VEGF-D mRNA is down-modulated either by beta-catenin mobilization from the cell membrane, by activation of the Wnt signaling pathway, or by transfection with the beta-catenin stable mutant. Down-modulation of beta-catenin by means of RNA interference showed an increase of VEGF-D mRNA steady state in fibroblasts. The beta-catenin-dependent decrease of VEGF-D mRNA is indirect and mainly due to reduced VEGF-D mRNA stability, as demonstrated by experiments of mRNA decay in the presence of transcription or translation inhibitors. By transient transfection of chimeric constructs carrying fusion of VEGF-D sequences under the control of the cytomegalovirus early promoter, we demonstrated that beta-catenin negative regulation is on the VEGF-D mRNA 3'-untranslated region. We mapped the VEGF-D mRNA-destabilizing element to a sequence, conserved between mouse and human VEGF-D, which contains an AU-rich element of group I. These results unveiled a new regulatory pathway for VEGF-D, which explains, at least in part, VEGF-D regulation in tumor progression.

3' Untranslated Regions↗
PubMed2001

cAMP stimulation of vasopressin and oxytocin release and regulation of vasopressin mRNA stability: role of auto-facilitation.

The effects of cycloheximide and actinomycin on 8-bromo-cAMP (8-Br-cAMP) stimulated vasopressin and oxytocin release from the posterior pituitary and vasopressin mRNA content of the supraoptic nucleus were studied with perifused explants of the hypothalamo-neurohypophyseal system. 8-Br-cAMP stimulated vasopressin and oxytocin release from the explant for up to 6 h. Inhibition of protein synthesis by cycloheximide completely suppressed the response to 8-Br-cAMP. When gene transcription was inhibited by actinomycin, vasopressin release was stimulated by 8-Br-cAMP for approximately 2 h, but the response was not sustained. Vasopressin mRNA content was not changed by 8-Br-cAMP in the absence or presence of cycloheximide, but it was significantly decreased by simultaneous exposure to 8-Br-cAMP and actinomycin. Actinomycin alone did not change vasopressin mRNA content. Since other studies have demonstrated that cAMP stimulates vasopressin gene transcription, and since vasopressin mRNA content reflects the balance between gene transcription and mRNA degradation, the effect of actinomycin and 8-Br-cAMP on vasopressin mRNA content suggests that 8-Br-cAMP also decreased vasopressin mRNA stability and thereby induced a rapid turnover of vasopressin mRNA. The effects of cycloheximide and actinomycin on vasopressin and oxytocin release suggest that ongoing protein synthesis is required for stimulation of hormone release. Since the posterior pituitary hormone stores are not depleted with a stimulus for release that is even more potent than cAMP, it is possible that cycloheximide and actinomycin depleted smaller pools of the peptides such as those responsible for intranuclear vasopressin and oxytocin release. Further evidence that intranuclear release of vasopressin and oxytocin is a prerequisite for cAMP stimulation of vasopressin and oxytocin release was obtained by demonstrating that d(CH2)5-D-Tyr(Me)VAVP, a potent combined V1a/V2/oxytocin receptor antagonist blocked stimulation of vasopressin and oxytocin release by 8-Br-cAMP.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Analysis of the altered mRNA stability (ams) gene from Escherichia coli. Nucleotide sequence, transcriptional analysis, and homology of its product to MRP3, a mitochondrial ribosomal protein from Neurospora crassa.

The product of the altered mRNA stability (ams) gene of Escherichia coli is involved in decay of mRNA. The complete nucleotide sequence of a 4-kilobase BamHI restriction fragment containing the ams coding sequence was determined. Transcription of the ams gene was analyzed by high resolution S1 mapping. A promoter was found with a homology score of 58% 361 nucleotides upstream from the start codon of ams. The ams structural gene consists of an open reading frame of 2,445 nucleotides. The protein predicted from this open reading frame has a molecular mass of 91,327 Da, which is significantly smaller than that determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Confirmation of the ams coding sequence was obtained by comparing the predicted amino acid sequence with that derived by amino-terminal analysis of gel-purified Ams protein. The predicted protein sequence of the ams gene was screened against translations of the GenBank DNA sequence data base. A homology of 18% over a region of 315 amino acids of the carboxyl terminus of the Ams product was found to MRP3, a mitochondrial ribosomal protein from Neurospora crassa. A smaller region of homology (29% in 86 residues) was found to the human U1 small nuclear ribonucleoparticle 70,000-Da protein.

Amino Acid Sequence↗
PubMed1995

Fatty acids regulate Thy-1 antigen mRNA stability in T lymphocyte precursors.

In this study, we report the effect of fatty acids on the Thy-1 antigen mRNA decay. Low serum and synthetic medium culture conditions were used to demonstrate that fatty acids, which are important metabolites involved as second messengers in signal transduction, also influence the steady-state mRNA level. Detailed analysis demonstrated that polyunsaturated lipids attached to bovine serum albumin, such as linoleic, linolenic, and arachidonic acids, modulate gene expression specifically in the S1A T lymphoma cell line by inducing a 3-5-fold increase in the steady-state Thy-1 mRNA level, concomitant with a twofold increase in cell surface expression. A similar modulation was observed in the immature CD4-CD8- T cell precursors but not in mature thymocytes. Nuclear run-on and transfection experiments indicated that the observed Thy-1 mRNA level is post-transcriptionally regulated and that the presence of the coding region is sufficient for this adaptive response. A mechanism without a requirement for protein kinase C activation, but involving Ca2+ entry, could account for this difference in Thy-1 mRNA stability.

Animals↗
PubMed2003

EhPgp5 mRNA stability is a regulatory event in the Entamoeba histolytica multidrug resistance phenotype.

The multidrug resistance (MDR) phenotype in Entamoeba histolytica is characterized by the overexpression of the EhPgp5 gene in trophozoites grown in high drug concentrations. Here we evaluated the role of EhPgp5 mRNA stability on MDR using actinomycin D. EhPgp5 mRNA from trophozoites growing without emetine had a half-life of 2.1 h, which augmented to 3.1 h in cells cultured with 90 microM and to 7.8 h with 225 microM emetine. Polyadenylation sites were detected at 118-, 156-, and 189-nucleotide (nt) positions of the EhPgp5 mRNA 3'-untranslated region. Interestingly, trophozoites grown with 225 microM emetine exhibited an extra polyadenylation site at 19 nt. The 3'-untranslated region sequence is AU-rich and has putative consensus sequences for RNA-binding proteins. We detected a RNA-protein complex in a region that contains a polypyrimidine tract (142-159 nt) and a cytoplasmic polyadenylation element (146-154 nt). A longer poly(A) tail in the EhPgp5 mRNA was seen in trophozoites grown with 225 microM emetine. Emetine stress may affect factors involved in mRNA turnover, including polyadenylation/deadenylation proteins, which could induce changes in the EhPgp5 mRNA half-life and poly(A) tail length. Novel evidence on mechanisms participating in E. histolytica MDR phenotype is provided.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

p53-independent elevation of p21 expression by PMA results from PKC-mediated mRNA stabilization.

The p21 (cip1/waf1) protein induces cell cycle arrest through inhibition of the activity of cdk (cyclin dependent kinase)/cyclin complexes. Expression of p21 is induced in a p53-dependent manner by DNA damage. p21 can also be induced independently of p53 by phorbol ester or okadaic acid. In this study, we have addressed the role of the PKC (protein kinase C) signaling pathway in the induction of p21 in response to PMA (phorbol myristate acetate) and okadaic acid. Levels of p21 (protein and mRNA) rapidly increased (within approximately 4 h) in U937 cells treated with PMA. The PKC-specific inhibitors RO 31-8220 and GF109203X down-regulated PMA or okadaic acid-induced p21 expression. Following persistent PKC activation, p21 mRNA levels remained elevated, indicating an enhanced stability of the mRNA. Using actinomycin D to measure mRNA stability and p21 promoter luciferase assays to measure activity, we provide evidence to support a role for the PKC signaling pathway in p21 mRNA stability. Thus, PKC regulates the amount of p21 in U937 cells at the level of mRNA accumulation and translation.

Cyclin-Dependent Kinase Inhibitor p21↗
PubMed2003

Maintenance and regulation of mRNA stability of the Saccharomyces cerevisiae OLE1 gene requires multiple elements within the transcript that act through translation-independent mechanisms.

The Saccharomyces cerevisiae OLE1 gene encodes a membrane-bound Delta-9 fatty acid desaturase, whose expression is regulated by unsaturated fatty acids through both transcriptional and mRNA stability controls. In fatty acid-free medium, the mRNA has a half-life of 10 +/- 1.5 min (basal stability) that drops to 2 +/- 1.5 min when cells are exposed to unsaturated fatty acids (regulated stability). A deletion analysis of elements within the transcript revealed that the sequences within the protein-coding region that encode transmembrane sequences and a part of the cytochrome b5 domain are essential for the basal stability of the transcript. Deletion of any of the three essential elements produced unstable transcripts and loss of regulated instability. By contrast, substitution of the 3'-untranslated region with that of the stable PGK1 gene did not affect the basal stability of the transcript and did not block regulated decay. Given that Ole1p is a membrane-bound protein whose activities are a major determinant of membrane fluidity, we asked whether membrane-associated translation of the protein was essential for basal and regulated stability. Insertion of stop codons within the transcript that blocked either translation of the entire protein or parts of the protein required for co-translation insertion of Ole1p had no effect. We conclude that the basal and regulated stability of the OLE1 transcript is resistant to the nonsense-mediated decay pathway and that the essential protein-encoding elements for basal stability act cooperatively as stabilizing sequences through RNA-protein interactions via a translation-independent mechanism.

3' Untranslated Regions↗
PubMed2003

Nitric oxide increases the decay of matrix metalloproteinase 9 mRNA by inhibiting the expression of mRNA-stabilizing factor HuR.

Dysregulation of extracellular matrix turnover is an important feature of many inflammatory processes. Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin-1 beta. We demonstrate that NO does strongly destabilize MMP-9 mRNA, since different luciferase reporter gene constructs containing the MMP-9 3' untranslated region (UTR) displayed significant reduced luciferase activity in response to the presence of NO. Moreover, by use of an in vitro degradation assay we found that the cytoplasmic fractions of NO-treated cells contained a higher capacity to degrade MMP-9 transcripts than those obtained from control cells. An RNA electrophoretic mobility shift assay demonstrated that three of four putative AU-rich elements present in the 3' UTR of MMP-9 were constitutively occupied by the mRNA-stabilizing factor HuR and that the RNA binding was strongly attenuated by the presence of NO. The addition of recombinant glutathione transferase-HuR prevented the rapid decay of MMP-9 mRNA, whereas the addition of a neutralizing anti-HuR antibody caused an acceleration of MMP-9 mRNA degradation. Furthermore, the expression of HuR mRNA and protein was significantly reduced by exogenously and endogenously produced NO. These inhibitory effects were mimicked by the cGMP analog 8-bromo-cGMP and reversed by LY-83583, an inhibitor of soluble guanylyl cyclase. These results demonstrate that NO acts in a cGMP-dependent mechanism to inhibit the expression level of HuR, thereby reducing the stability of MMP-9 mRNA.

3' Untranslated Regions↗
PubMed1997

Modulation of the hepatic expression of the estrogen-regulated mRNA stabilizing factor by estrogenic and antiestrogenic nonsteroidal xenobiotics.

Estrogen-mediated accumulation of apolipoprotein II (apoII) mRNA in the avian liver is due, in part, to its stabilization. This stabilization appears to be due to the estrogen-regulated mRNA stabilizing factor (E-RmRNASF) that is expressed in response to estrogen. The E-RmRNASF protects the mRNA from targeted endonucleolytic degradation (Ratnasabapathy, Cell Mol Biol Res 41: 583-594, 1995). To determine whether certain environmental xenobiotics altered the expression of the gene encoding E-RmRNASF by mimicking estrogen, roosters were given estrogen, tamoxifen, clomiphene, hexachlorophene, lindane, rotenone, chlordecone, dichlorodiphenyltrichloroethane (DDT); Araclor, methoxychlor, dieldrin, toxaphene, or bisphenol-A parenterally. Uniformly radiolabeled, capped, and polyadenylated apoII mRNA, incubated in vitro in the presence of liver cytosolic extracts from birds that received estrogen, tamoxifen, hexachlorophene, chlordecone, or Araclor, remained stable, indicating that these agents were estrogenic and stimulated the expression of E-RmRNASF. However, the same mRNA was degraded in similar extracts from control roosters and those treated with clomiphene, DDT, methoxychlor, dieldrin, rotenone, toxaphene, lindane, or bisphenol-A. To determine whether the latter agents were antiestrogenic, roosters were given a 1:5 molar combination of estrogen and each of the xenobiotics. ApoII mRNA showed degradation in liver extracts from roosters that received clomiphene, toxaphene, or bisphenol-A, indicating that these agents prevented estrogenic stimulation of expression of the E-RmRNASF and were antiestrogenic. However, the rest of the xenobiotics failed to antagonize estrogenic stimulation of E-RmRNASF gene expression. These results set a precedent in showing the estrogenic and antiestrogenic effects in vivo of environmental xenobiotics on the expression of a regulatory protein involved in estrogen-mediated mRNA stabilization.

Animals↗
PubMed1994

Cellular localization of RNA14p and RNA15p, two yeast proteins involved in mRNA stability.

RNA14 and RNA15 were originally identified by temperature-sensitive mutations that cause a rapid decrease in poly(A)-tail length and overall mRNA levels at the restrictive temperature. We have raised antibodies to the RNA14 and RNA15 proteins, and used subcellular fractionation and immunofluorescence to localize these proteins within the yeast cell. RNA14p is a 73 kDa protein found in both the nucleus and the cytoplasm, whilst RNA15p is a 42 kDa protein detected only in the nucleus. The observation that both proteins are found in the nucleus is in agreement with previous genetic data which suggest an interaction between RNA14p and RNA15p. Also the joint nuclear localization is consistent with the biochemical data suggesting a role in polyadenylation. The detection of significant amounts of RNA14p in the cytoplasm opens the possibility of a second function for this protein, either in cytoplasmic regulation of mRNA deadenylation or, more interestingly, in mRNA stability.

Cell Fractionation↗