Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “mRNA stability”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,063 records · Page 59Linked to original sources
PubMed1994

Interplay of two functionally and structurally distinct domains of the c-fos AU-rich element specifies its mRNA-destabilizing function.

AU-rich elements (ARE) in the 3' untranslated region of many highly labile mRNAs for proto-oncogenes, lymphokines, and cytokines can act as an RNA-destabilizing element. The absence of a clear understanding of the key sequence and structural features of the ARE that are required for its destabilizing function has precluded the further elucidation of its mode of action and the basis of its specificity. Combining extensive mutagenesis of the c-fos ARE with in vivo analysis of mRNA stability, we were able to identify mutations that exhibited kinetic phenotypes consistent with the biphasic decay characteristic of a two-step mechanism: accelerated poly(A) shortening and subsequent decay of the transcribed portion of the mRNA. These mutations, which affected either an individual step or both steps, all changed the mRNA stability. Our experiments further revealed the existence of two structurally distinct and functionally interdependent domains that constitute the c-fos ARE. Domain I, which is located within the 5' 49-nucleotide segment of the ARE and contains the three AUUUA motifs, can function as an RNA destabilizer by itself. It forms the essential core unit necessary for the ARE-destabilizing function. Domain II is a 20-nucleotide U-rich sequence which is located within the 3' part of the c-fos ARE. Although it alone can not act as an RNA destabilizer, this domain serves two critical roles: (i) its presence enhances the destabilizing ability of domain I by accelerating the deadenylation step, and (ii) it has a novel capacity of buffering decay-impeding effects exerted by mutations introduced within domain I. A model is proposed to explain how these critical structural features may be involved in the c-fos ARE-directed mRNA decay pathway. These findings have important implications for furthering our understanding of the molecular basis of differential mRNA decay mediated by different AREs.

3T3 Cells↗
PubMed2005

Oncostatin M causes VEGF release from human airway smooth muscle: synergy with IL-1beta.

Vascular endothelial growth factor (VEGF), a potent angiogenesis factor, likely contributes to airway remodeling in asthma. We sought to examine the effects and mechanism of action of IL-6 family cytokines on VEGF release from human airway smooth muscle (HASM) cells. Oncostatin M (OSM), but not other IL-6 family cytokines, increased VEGF release, and IL-1beta enhanced OSM-induced VEGF release. OSM increased VEGF mRNA expression and VEGF promoter activity, whereas IL-1beta had no effect. IL-1beta did not augment the effects of OSM on VEGF promoter activity but did augment OSM-induced VEGF mRNA expression and mRNA stability. The STAT3 inhibitor piceatannol decreased both OSM-induced VEGF release and synergy between OSM and IL-1beta, without affecting responses to IL-1beta alone. Piceatannol also inhibited OSM-induced VEGF mRNA expression. In contrast, inhibitors of MAPK pathway had no effect on OSM or OSM plus IL-1beta-induced VEGF release. OSM increased type 1 IL-1 receptor (IL-1R1) mRNA expression, as measured by real-time PCR, and piceatannol attenuated this response. Consistent with the increase in IL-1R1 expression, OSM markedly augmented IL-1beta-induced VEGF, MCP-1, and IL-6 release. In summary, our data indicate OSM causes VEGF expression in HASM cells by a transcriptional mechanism involving STAT3. IL-1beta also synergizes with OSM to increase VEGF release, likely as a result of effects of IL-1beta on VEGF mRNA stability as well as effects of OSM on IL-1R1 expression. This is the first description of a role for OSM on IL-1R1 expression in any cell type. OSM may contribute to airway remodeling observed in chronic airway disease.

Cells, Cultured↗
PubMed1992

Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis.

Understanding the regulation of hepatic glucose metabolism had its foundation in the elucidation of several pathways, but recent advances have come from the application of molecular genetics. Five years ago little was known about the primary structure of the key regulatory enzymes. Since then, the primary sequence of liver GK, 6-PF-1-K, Fru-1,6-P2ase, PK, PEPCK, and 6-PF-2-K/Fru-2,6-P2ase have been derived from cDNA sequences and/or determined by direct protein sequencing. This has provided new insights into the molecular mechanisms of catalysis and the regulation of these enzymes by covalent modification. Isolation of the cDNAs for these enzymes also has allowed for the quantitation of specific mRNAs and permitted analysis of hormonal control of specific gene expression. The genes for these enzymes have been isolated and sequenced, and their promoter regions are being identified and characterized. Hormone response elements have been delineated in several of the promoters. The promoter regions for 6-PF-2-K/Fru-2,6-P2ase and Fru-1,6-P2ase have also been identified, and future research will focus on the elucidation of the mechanisms whereby hormones regulate the expression of these genes. A number of generalizations can be made about the regulation of gene expression of glycolytic/gluconeogenic enzymes. First, there is coordinate hormonal regulation of gene expression and these effects are consonant with their physiologic actions. Insulin induces the mRNAs that encode glycolytic enzymes and represses the mRNAs that encode gluconeogenic enzymes; cAMP has opposite effects. Both can increase or decrease transcription. Whereas insulin and cAMP affect all of these mRNAs, glucocorticoids appear to have a more restricted action. Second, transcriptional and posttranscriptional regulatory mechanisms are involved. The synthesis of all of the mRNAs discussed is regulated by hormones. Relatively little is known about how mRNA stability is regulated in general, but it is clear that PEPCK mRNA is stabilized by agents that increase the rate of transcription of the gene. Under appropriate metabolic signals this dual control of mRNA synthesis and stability provides for a long-term increase in PEPCK mRNA and protein. Studies with PK mRNA are less direct, but suggest a similar dual mechanism. It will be interesting to see whether multilevel regulation is restricted to these two mRNAs, both of which are involved in the same substrate cycle, or whether the stability of other mRNAs involved in hepatic glucose metabolism is also affected. Third, glucose appears to be important in the regulation of these hepatic genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2006

Neutrophil elastase-initiated EGFR/MEK/ERK signaling counteracts stabilizing effect of autocrine TGF-beta on tropoelastin mRNA in lung fibroblasts.

Neutrophil elastase (NE) plays an important role in emphysema, a pulmonary disease associated with excessive elastolysis and ineffective repair of interstitial elastin. Besides its direct elastolytic activity, NE releases soluble epidermal growth factor receptor (EGFR) ligands and initiates EGFR/MEK/ERK signaling to downregulate tropoelastin mRNA in neonatal rat lung fibroblasts (DiCamillo SJ, Carreras I, Panchenko MV, Stone PJ, Nugent MA, Foster JA, and Panchenko MP. J Biol Chem 277: 18938-18946, 2002). We now report that NE downregulates tropoelastin mRNA in the rat fetal lung fibroblast line RFL-6. The tropoelastin mRNA downregulation is preceded by release of EGF-like and TGF-alpha-like polypeptides and requires EGFR/MEK/ERK signaling, because it is prevented by the EGFR inhibitor AG1478 and the MEK/ERK uncoupler U0126. Tropoelastin expression in RFL-6 fibroblasts is governed by autocrine TGF-beta signaling, because TGF-beta type I receptor kinase inhibitor or TGF-beta neutralizing antibody dramatically decreases tropoelastin mRNA and protein levels. Half-life of tropoelastin mRNA in RFL-6 cells is >24 h, but it is decreased to approximately 8 h by addition of TGF-beta neutralizing antibody, EGF, TGF-alpha, or NE. Tropoelastin mRNA destabilization by NE, EGF, or TGF-alpha is abolished by AG1478 or U0126. EGF-dependent tropoelastin mRNA downregulation is reversed upon ligand withdrawal, whereas chronic EGF treatment leads to persistent downregulation of tropoelastin mRNA and protein levels and decreases insoluble elastin deposition. We conclude that NE-initiated EGFR/MEK/ERK signaling cascade overrides the autocrine TGF-beta signaling on tropoelastin mRNA stability and, therefore, decreases the elastogenic response in RFL-6 fibroblasts. We hypothesize that persistent EGFR/MEK/ERK signaling could impede the TGF-beta-induced elastogenesis/elastin repair in the chronically inflamed, elastase/anti-elastase imbalanced lung in emphysema.

Animals↗
PubMed1993

Structure and regulation of expression of the acetylcholinesterase gene.

Acetylcholinesterase, an enzyme essential for the termination of the action of acetylcholine, is encoded by a single gene. Alternative mRNA processing gives rise to the expression of enzyme forms with three distinct carboxyl-termini. These structural differences govern the cellular disposition of the expressed enzyme but do not influence catalytic activity. Alternative polyadenylation signals give rise to distinct 3' non-coding regions which are likely to affect mRNA stability. Alternative splicing also occurs at the 5' end of the gene where two promoter regions can be identified. Hence, regulation of expression of the gene occurs at 3 levels, transcriptional through alternative promoters, translational by affecting mRNA stability and processing of distinct mRNAs and post-translationally by giving rise to distinct peptide chains which are processed differently. Recombinant DNA studies have also been extended to modifying protein structure through site-specific mutagenesis and studying the function of the mutant enzymes.

Acetylcholinesterase↗
PubMed1995

Regulated specific protein binding to a conserved region of the 3'-untranslated region of thyrotropin beta-subunit mRNA.

Thyroid hormone (T3) regulates the expression of rat TSH beta-subunit (TSH beta) mRNA, in part, at the posttranscriptional level, by reducing the half-life of TSH beta mRNA. The mechanism(s) mediating this alteration in mRNA stability are unknown, but previous work indicates that labile protein(s) are involved. The majority of cis-acting elements identified to date that have been implicated in the regulated destabilization of mRNAs have been located in the 3'-untranslated region (3'-UTR) of the mRNA. The 3'-UTR of rat, murine, and human TSH beta mRNA is highly conserved, and within this region is a 12-nucleotide consensus sequence, which is shared by the 3'-UTR of several other genes with unstable mRNAs. We reasoned that this homologous region could represent a binding motif for specific trans-acting RNA-binding protein(s), and that identification and characterization of such trans-acting factor(s) may provide critical insight into the mechanisms underlying T3-induced changes in TSH beta mRNA stability. Utilizing the RNA electrophoretic mobility shift assay and analysis of UV cross-linked RNA-protein complexes, a cytoplasmic trans-acting factor of approximately 80-85 kilodaltons was identified from rat pituitaries and several cell lines that binds in a sequence-specific manner to the 3'-UTR of rat TSH beta mRNA. Using competitive antisense oligonucleotides, the predominant binding site was mapped to the first 41 nucleotides of the 3'-UTR, which includes the consensus region. However, sequence upstream of the consensus was also shown to be important for binding. Using RNA electrophoretic mobility shift assay, two mRNAs containing sequence homology with the consensus region, c-erbA alpha-2 and a rat ferritin pseudogene, were shown to specifically compete with rat TSH beta mRNA for binding of this factor. Remarkably, the binding activity of this factor was regulated positively by T3 within 4 h, but only with rat pituitary extracts. These data suggest that in addition to binding rat TSH beta mRNA in a sequence-specific and T3-regulated manner, this novel trans-acting RNA-binding protein may also bind to other cytoplasmic mRNAs involved in diverse intracellular processes.

Animals↗
PubMed2001

Transcriptional up-regulation of p27(Kip1) during contact-induced growth arrest in vascular endothelial cells.

By plating porcine aortic endothelial cells at two different densities and thereby inducing two different time courses of contact-induced growth arrest, the temporal correlation between p27(Kip1) expression and cell cycle progression was investigated. When the quiescent cells were replated, they synchronously entered S phase with a peak at 20 h in both cases, while the cells plated at 25 and 80% of confluent densities exited the cell cycle by 96 and 48 h, respectively. Nuclear p27(Kip1) disappeared when the cells reentered the cell cycle and then recovered when the cells exited the cell cycle. The change in p27(Kip1) was associated with a concomitant change in Kip1 mRNA. The p27(Kip1) degradation activity did not increase in the cells reentering the cell cycle, nor did it decrease in the cells exiting the cell cycle. The Kip1 mRNA stability decreased in the growing cells and increased in the cells exiting the cell cycle and at confluence. A nuclear run-on assay revealed a close correlation between the Kip1 transcriptional activity and the level of Kip1 mRNA. We conclude that the cell-cell contact up-regulated the Kip1 gene transcription and increased the Kip1 mRNA stability, which was related to the recovery of p27(Kip1) protein during contact-induced growth arrest in endothelial cells.

Animals↗
PubMed1991

Modulation of complement gene expression by glucocorticoids.

The addition of dexamethasone, prednisolone or cortisol (in order of efficacy) to human monocytes in culture produced dose-related increases in the synthesis rates of the complement components C1 inhibitor (C1-inh), factor B (B) and C2. In contrast, concentrations of C3 and lysozyme in the culture supernatants were decreased. Indomethacin stimulated synthesis of C1-inh, C2 and B, but had little effect on synthesis of C3 or lysozyme. The simultaneous addition of cycloheximide (2.5 micrograms/ml) abrogated the effects of dexamethasone on synthesis of C2, B and C1-inh, but the effect of indomethacin on the synthesis of these components was unchanged. These data suggest that protein synthesis is required for the effects of glucocorticoids on the synthesis of C2, B and C1-inh to occur. Dexamethasone and indomethacin increased the abundances of C1-inh mRNA, B mRNA and C2 mRNA in parallel with changes in the synthesis rates of these proteins. The changes in mRNA abundance were not transcriptional, but were shown to be due to increased mRNA stability. In contrast, dexamethasone decreased the expression of C3 and lysozyme by decreasing the rate of transcription of these genes. Indomethacin had no effect on transcription of the C3 and lysozyme genes. The half-lives of C3 mRNA, lysozyme mRNA and actin mRNA were not altered by dexamethasone or indomethacin. It is concluded that the effects of glucocorticoids on monocyte synthesis of C2, B and C1-inh are due to increased mRNA stability and may be related to inhibition of prostaglandin synthesis, as these effects are similar to those produced by indomethacin. The effects of dexamethasone on the synthesis of C3 and lysozyme differ from those on C2, B and C1-inh as they depend upon a decrease in gene transcription, which is not affected by indomethacin.

Cells, Cultured↗
PubMed1998

Interleukin (IL)-1 and IL-4 synergistically stimulate NF-IL6 activity and IL-6 production in human mesangial cells.

BACKGROUND: While interleukin (IL)-4 inhibits pro-inflammatory cytokine expression by human monocytes, we have observed that it potentiates IL-6 production by IL-1-activated human mesangial cells (MC). To study the mechanism of this cell-type specific interaction between IL-1 and IL-4 in MC, we examined the effect of both cytokines on the activities of nuclear factor kappa B (NF-kappa B) and nuclear IL-6 NL-IL 6), transcription factors that are essential for IL-6 gene expression. METHODS: We evaluated IL-6 synthesis, mRNA expression, and mRNA stability by ELISA, Northern analysis, and the actinomycin D method, respectively. Activities of NF-kappa B and NF-IL 6 were analyzed by gel shift assay. RESULTS: IL-4 augmented the IL-1 stimulated IL-6 mRNA levels by about threefold without altering mRNA stability. IL-1 treatment rapidly induced the binding activity of NF-kappa B. In contrast, IL-4 did not affect basal and IL-1-induced NF-kappa B activities. Both IL-1 and IL-4 stimulated NF-IL6 activity as early as 30 minutes after treatment. When MC were treated with both cytokines together, marked activation of NF-IL6 was observed at five hours. CONCLUSIONS: These results suggest that simultaneous activation of NF-kappa B and NF-IL6 is essential for IL-6 gene expression and that IL-1 and IL-4 cooperatively stimulate MC IL-6 production through their synergistic activation of NF-IL6.

CCAAT-Enhancer-Binding Proteins↗
PubMed2005

Transcriptional and posttranscriptional regulation of the plasminogen activator system.

The core protein components of the plasminogen activator (PA) system are two plasminogen activators, two plasminogen activator inhibitors and a urokinase type plasminogen activator-specific cell surface receptor. Various types of biological regulation are exerted through the interplay of these components mutually and with extracellular matrix proteins and cell membrane proteins, with or without involving proteolytic activity. Reflecting these diverse biological roles, the level and activity of each component of the PA system is under the control of a variety of regulatory mechanisms. The expression level of a protein reflects the level of the corresponding mRNA, which is essentially the net result of de novo synthesis, i.e. transcription, and degradation. Many recent studies have shown that the regulation of mRNA stability is dynamic and cell specific. Accordingly, we are learning that the mRNAs of the PA system are also the subject of diverse regulatory mechanisms. In this short review, we summarize current understanding of the transcriptional and mRNA-stability regulation of the PA system.

Adipocytes↗
PubMed1998

Characterization of ribonucleoprotein complexes and their binding sites on the neurofilament light subunit mRNA.

Levels of neurofilament (NF) gene expression are important determinants of basic neuronal properties, but overexpression can lead to motoneuron degeneration in transgenic mice. In a companion study (Cañete-Soler, R., Schwartz, M. L., Hua, Y., and Schlaepfer, W. W. (1998) J. Biol. Chem. 273, 12650-12654), we show that levels of NF expression are regulated by altering mRNA stability and that stability determinants are present in the 3'-coding region (3'-CR) and 3'-untranslated region (3'-UTR) of the NF light subunit (NF-L) transcript. This study characterizes the ribonucleoprotein complexes that bind to the NF-L mRNA when cytoplasmic brain extracts are incubated with radioactive probes. Gel retardation assays reveal ribonucleoprotein complexes that are selectively competed with poly(C) or poly(U))/poly(A) homoribopolymers and are referred to as C-binding and U/A-binding complexes, respectively. The C-binding complex forms on the proximal 45 nucleotides of 3'-UTR, but its assembly is markedly enhanced by 23 nucleotides of flanking 3'-CR sequence. U/A-binding complexes form at multiple binding sites in the 3'-CR and 3'-UTR. A pattern of reciprocal binding suggests that the C-binding and U/A-binding complexes interact and may compete for common components or binding sites. Cross-linking studies reveal unique polypeptides in the C-binding and U/A-binding complexes. The findings provide the basis for probing mechanisms regulating NF-L mRNA stability and the relationship between NF overexpression and motoneuron degeneration in transgenic mice.

Animals↗
PubMed1988

The stability of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells varies with growth rate.

The decay rates of eucaryotic elongation factor Tu (eEF-Tu) mRNA and eucaryotic initiation factor 4A (eIF-4A) mRNA in Friend erythroleukemia (FEL) cells were determined under several different growth conditions. In FEL cells which were no longer actively dividing (stationary phase), eEF-Tu mRNA was found to be rather stable, with a t1/2 of about 24 h. In rapidly growing FEL cells eEF-Tu mRNA was considerably less stable, with a t1/2 of about 9 h. In both cases a single rate of mRNA decay was observed. However, when stationary-phase cells resumed growth after treatment with fresh medium, we observed that eEF-Tu mRNA decay followed a biphasic process. The faster of the two decay rates involved approximately 50% of the eEF-Tu mRNA and had a t1/2 of about 1 h. The decay rates for eIF-4A (t1/2 = 2 h) and total poly(A)+ RNA (t1/2 = 3 h) were unaffected by changes in growth conditions. The t1/2 for polysomal eEF-Tu mRNA was found to be about 8 h when stationary FEL cells were treated with fresh medium. Previous work in this laboratory has shown (T. R. Rao and L. I. Slobin, Mol. Cell. Biol. 7:687-697, 1987) that when FEL cells are allowed to grow to stationary phase, approximately 60% of the mRNA for eEF-Tu is found in a nontranslating postpolysomal messenger ribonucleoprotein (mRNP) particle. eEF-Tu mRNP was rapidly cleared from stationary cells after treatment with fresh medium. The data presented in this report indicate that the stability of eEF-Tu mRNP is rapidly altered and the particle is targeted for degradation when stationary FEL cells resume growth.

Cell Division↗
PubMedDate not supplied

Transcriptional regulation of cyclo-oxygenase expression: three pillars of control.

Blocking cyclo-oxygenase (COX) isoform activities with non-steroidal anti-inflammatory drugs (NSAIDS) is widely employed in the treatment of arthritis. These agents also hold great promise in the context of pre and post-neoplastic diseases such as colorectal cancer (CRC). Nevertheless, issues of isoform specificity and delivery necessitate the exploration of other strategies to specifically block expression of the COX genes. Approaches that target gene transcription may complement enzyme inhibition. Thus, understanding the regulation of COX isoform transcription may improve the specific inhibition of expression. Three tiers of transcriptional regulation are evident: initiation, alternative splicing and messenger RNA stability. Transcription factors that activate COX-2 expression are elevated in certain disease states and emergency responses such as infection and are therefore potential targets. These factors include C/EBP-beta, phospho- CREB, NF-IL6, AP1, NFkB, and TCF-4/LEF-1. In this review we highlight another factor, c-MYB as a key COX-2 regulator in CRC. Alternative exon usage is another tier of regulation that has not received much attention. For instance, COX-1 splice variants (also known as COX-3 and PCOX-1a) may broaden the spectrum of COX activities in disease. Similarly, whilst mRNA stability is clearly modulated by steroids in the case of COX-2, the wider implications of targeting mRNA stability have not been afforded the same attention. Finally, it seems that some NSAIDS exert part of their action directly on COX-2 transcriptional regulation explaining why such agents display greater effects on this isoform than enzyme inhibition data would suggest.

Animals↗
PubMed2000

Increased stability of macrophage migration inhibitory factor (MIF) in DU-145 prostate cancer cells.

Macrophage migration inhibitory factor (MIF) has been localized to the glandular epithelium of the prostate and stimulates the in vitro growth of prostate epithelial cells. [35S]Methionine labeling of MIF protein was used to determine if prostate cells synthesize and secrete this cytokine. The results demonstrated that the DU-145 prostate cancer cells secrete about twice the amount of a more stable protein compared with normal prostate epithelial cells. To investigate if differences in MIF mRNA levels account for the differences in MIF protein secreted by these cells, mRNA stability was analyzed by [3H]uridine incorporation. Following a 12-h pulse, DU-145 cells were found to contain four times the amount of [3H]uridine-labeled MIF mRNA, and this message exhibited a longer half-life than the message found in normal cells (33 h and 19 h, respectively). Nuclear run-on experiments confirmed that the MIF gene is transcribed at a greater rate (1.8-fold) in the DU-145 prostate cancer cells. This study documents, for the first time, that human prostate epithelial cells synthesize and secrete this cytokine. These results indicate that the increased levels of MIF found in prostate cancer cells is likely due to the increased protein and mRNA stability as exhibited by DU-145 cells.

Adenocarcinoma↗
PubMed1996

Promoter-dependent and -independent activation of insulin-like growth factor binding protein-5 gene expression by prostaglandin E2 in primary rat osteoblasts.

Insulin-like growth factor (IGF) action is mediated by high affinity cell surface IGF receptors and modulated by a family of secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of six IGFBPs characterized to date, uniquely potentiates the anabolic actions of IGF-I for skeletal cells. In osteoblasts, IGFBP-5 production is stimulated by prostaglandin E2 (PGE2), a local factor that mediates certain effects induced by parathyroid hormone, cytokines such as interleukin-1 and transforming growth factor-beta, and mechanical strain. In this study, we show that transcriptional and post-transcriptional events initiated by PGE2 collaborate to enhance IGFBP-5 gene expression in primary fetal rat osteoblast cultures. PGE2 treatment stimulated up to a 7-fold rise in steady-state levels of IGFBP-5 mRNA throughout 32 h of incubation. Analysis of nascent IGFBP-5 mRNA suggested that PGE2 had only a modest stimulatory effect on IGFBP-5 gene transcription, and transient transfection studies with IGFBP-5 promoter-reporter genes confirmed that PGE2 enhanced promoter activity by approximately 2-fold. Similar stimulatory effects were seen with forskolin. A DNA fragment with only 51 base pairs of the 5'-flanking sequence retained hormonal responsiveness, which may be mediated by a binding site for transcription factor AP-2 located at positions -44 to -36 in the proximal IGFBP-5 promoter. Incubation of osteoblasts with the mRNA transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that PGE2 enhanced IGFBP-5 mRNA stability by 2-fold, increasing the t1/2 from 9 to 18 h. The effects of PGE2 on steady-state IGFBP-5 transcripts were abrogated by preincubating cells with cycloheximide, indicating that the effects of PGE2 on both gene transcription and mRNA stability required ongoing protein synthesis. Therefore, both promoter-dependent and -independent pathways converge to enhance IGFBP-5 gene expression in response to PGE2 in osteoblasts.

Animals↗
PubMed2000

The E1E4 protein of human papillomavirus type 16 associates with a putative RNA helicase through sequences in its C terminus.

Human papillomavirus type 16 (HPV16) infects cervical epithelium and is associated with the majority of cervical cancers. The E1E4 protein of HPV16 but not those of HPV1 or HPV6 was found to associate with a novel member of the DEAD box protein family of RNA helicases through sequences in its C terminus. This protein, termed E4-DBP (E4-DEAD box protein), has a molecular weight of 66,000 (66K) and can shuttle between the nucleus and the cytoplasm. It binds to RNA in vitro, including the major HPV16 late transcript (E1E4. L1), and has an RNA-independent ATPase activity which can be partially inhibited by E1E4. E4-DBP was detectable in the cytoplasm of cells expressing HPV16 E1E4 (in vivo and in vitro) and could be immunoprecipitated as an E1E4 complex from cervical epithelial cell lines. In cell lines lacking cytoplasmic intermediate filaments, loss of the leucine cluster-cytoplasmic anchor region of HPV16 E1wedgeE4 resulted in both proteins colocalizing exclusively to the nucleoli. Two additional HPV16 E1E4-binding proteins, of 80K and 50K, were identified in pull-down experiments but were not recognized by antibodies to E4-DBP or the conserved DEAD box motif. Sequence analysis of E4-DBP revealed homology in its E4-binding region with three Escherichia coli DEAD box proteins involved in the regulation of mRNA stability and degradation (RhlB, SrmB, and DeaD) and with the Rrp3 protein of Saccharomyces cerevisiae, which is involved in ribosome biogenesis. The synthesis of HPV16 coat proteins occurs after E1E4 expression and genome amplification and is regulated at the level of mRNA stability and translation. Identification of E4-DBP as an HPV16 E1E4-associated protein indicates a possible role for E1E4 in virus synthesis.

Cell Nucleus↗
PubMed1997

Stabilization of myelin mRNAs as measured in a brain slice system.

The stabilization and destabilization of myelin mRNA is increasingly recognized as a major control point in regulating myelin gene expression. A brain slice system was developed and characterized to study mRNA stability in actively myelinating oligodendrocytes. The mRNA half-life of a major CNS myelin protein, proteolipid protein (PLP), was measured to be 5 hr. The half-life of another CNS myelin protein mRNA, myelin basic protein (MBP), was measured to be greater than 12 hr. A long half-life for MBP mRNA is consistent with MBP mRNA being stable long enough to be translocated to the myelin internode where it is then translated. Using semi-quantitative reverse transcriptase-PCR, it was determined that there was no differential stabilization between the two major PLP mRNA isoforms, PLP and DM20. It was also determined that protein synthesis was required for the specific stabilization of PLP/DM20 mRNAs. Inasmuch as PLP is a major structural protein of the CNS myelin, the PLP/DM20 mRNAs have relatively short half-lives. However, the PLP/DM20 mRNAs half-lives may be increased by the action of trans-acting factors that are themselves very labile.

Animals↗
PubMed1997

Regulation of interleukin-10 gene expression: possible mechanisms accounting for its upregulation and for maturational differences in its expression by blood mononuclear cells.

Interleukin-10 (IL-10) downmodulates phagocytic immune responses and accentuates humoral responses. Human neonates exhibit broad immune deficits that parallel actions of IL-10. We postulated that IL-10 production would be diminished in neonatal blood cells. We found that IL-10 production by lipopolysaccharide-stimulated peripheral blood mononuclear cells (PBMNCs) in vitro was greater by adult cells than by term cells and preterm cells. Additional studies were undertaken to identify mechanisms responsible for the developmental differences in IL-10 gene expression. IL-10 transcription was present in freshly isolated adult and neonatal cells in the absence of detectable levels of transcript. Transcription rates were not different between adult and neonatal cells. IL-10 transcripts were approximately 40% more abundant in adult cells than in term cells and were consistent with differences in secreted protein; however, no differences were noted in mRNA stability. IL-10 half-life was 60 minutes for both adult and term PBMNCs. We conclude that up-regulation of IL-10 gene expression in PBMNCs is modulated at the post-transcriptional level, that IL-10 protein production and mRNA content are greater in activated cells from adults compared with those from neonates, and that maturational differences in IL-10 expression are not due to differences in transcription rate or mRNA stability. Maturational differences in IL-10 expression might be due to differences in subpopulations of cytokine-producing cells or differences in nucleo-cytoplasmic transport.

Adult↗