ABSTRACT
Mammalian ribonucleotide reductase is a highly regulated activity essential for DNA synthesis and repair. The activity and message levels of the enzyme are elevated in cells treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, and this appears to be mediated through specific cis elements in the 3'-untranslated region of the R1 and R2 mRNAs that interact with R1 and R2 binding proteins called R1BP and R2BP, respectively. Hydroxyurea-resistant cells with increased R1 and R2 message levels were observed to have increased R1 and R2 message half-lives. This was accompanied by alterations in R1 and R2 3'-untranslated region cis-trans interactions, as judged by band shift and UV cross-linking assays, in which R1BP and R2BP binding was markedly reduced. This first description of mutant mammalian cells altered in message stability regulatory determinants indicates another mechanism for acquiring resistance to an antitumor agent. Furthermore, the present study strongly supports the concept that R1BP and R2BP are important general regulators of ribonucleotide reductase message stability and act as message destabilizing factors.
Subject(s)
Antineoplastic Agents/pharmacology , Gene Expression Regulation/drug effects , Hydroxyurea/pharmacology , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Ribonucleotide Reductases/genetics , Animals , Base Sequence , Binding Sites , Carcinogens/pharmacology , Drug Resistance, Neoplasm , In Vitro Techniques , L Cells , Mice , Molecular Sequence Data , Regulatory Sequences, Nucleic Acid , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
The transforming growth factor, TGF-beta(1), has been found to be increased in the central nervous system of Alzheimer's disease (AD) patients, elevates amyloid precursor protein (APP) mRNA levels in rat primary astrocytes, and may initiate or promote the deposition of amyloid-beta (Abeta) peptide in AD. Excess APP production in AD, which potentially leads to amyloidogenesis, is in part due to over expression of APP mRNA. The production of APP in a normal human cell line in contrast to transformed or animal cells provides a meaningful model to study the regulation of APP gene expression by cytokines that promotes amyloidogenesis. Here, we report that TGF-beta(1) treatment of human astrocytes markedly elevated APP mRNA levels, and also increased the half-life of APP message by at least five-fold. Under this condition, as detected by mobility shift and UV cross-linking analysis, a novel 68 kDa RNA-protein complex was formed, involving an 81 nucleotide (nt) fragment within the 3'-untranslated region (UTR), but not the 5'-UTR and coding region of APP mRNA. Insertion of the 3'-UTR onto the chloramphenicol acetyl transferase (CAT) mRNA conferred TGF-beta(1) mediated mRNA stability in transfected human astrocytes. On the other hand, the same insert carrying a deletion of the APP mRNA cis-element fragment had no effect on CAT mRNA stability. A model of APP mRNA regulation is presented in which TGF-beta(1) induced stabilization of APP message involves the binding activity of a 68 kDa RNA-protein complex within the 3'-UTR, which is likely linked to a reduction in the rate of APP mRNA decay.
Subject(s)
Amyloid beta-Protein Precursor/genetics , Astrocytes/metabolism , Brain/metabolism , Gene Expression Regulation/drug effects , RNA, Messenger/metabolism , Transcription, Genetic/drug effects , Transforming Growth Factor beta/pharmacology , 3' Untranslated Regions/genetics , Animals , Astrocytes/drug effects , Cell Line , Chloramphenicol O-Acetyltransferase/genetics , Humans , Kinetics , RNA, Messenger/genetics , Rats , Recombinant Fusion Proteins/biosynthesis , Templates, Genetic , TransfectionABSTRACT
Wild-type Friend mouse erythroleukaemia cells (clone 707) were compared with adenine phosphoribosyltransferase (APRT)-deficient mutant subclones (707DAP8 and 707DAP10) for sensitivity to cell killing and mutagenesis by ethyl methanesulphonate (EMS) and methyl methanesulphonate (MMS). Cells were exposed to 0-300 micrograms/ml EMS and to 0-20 micrograms/ml MMS for a period of 16 h. A slight difference was found between wild-type cells and the two APRT-deficient subclones in terms of sensitivity to cell killing by both mutagens. The APRT-deficient subclones were, however, significantly more sensitive than wild-type cells to mutagenesis to 5-bromo-2-deoxyuridine resistance and 6-thioguanine resistance by EMS and MMS. The APRT-deficient subclones were found to have significantly decreased levels of dATP and dTTP nucleotides and decreased levels of all four ribonucleoside triphosphates (ATP, GTP, CTP and UTP) relative to wild-type cells. Wild-type Friend cells were found to have insignificant levels O6-methylguanine-DNA methyl transferase and it is suggested that the increased mutagen sensitivity of APRT-deficient cells may be due to imbalance of deoxyribonucleoside triphosphate pools during DNA excision-repair processes, or more probably due to deficiency of ATP for ATP-dependent DNA excision-repair enzymes.
Subject(s)
Alkylating Agents/pharmacology , Friend murine leukemia virus , Leukemia, Erythroblastic, Acute/genetics , Mutagenesis/drug effects , Nucleotides/analysis , Animals , Cell Line , Deoxyribonucleotides/analysis , Dose-Response Relationship, Drug , Ethyl Methanesulfonate/pharmacology , In Vitro Techniques , Methyl Methanesulfonate/pharmacology , Methyltransferases/analysis , Mice , Ribonucleosides/analysisABSTRACT
AIMS: To examine the expression and localization of osteopontin (OPN), a secreted phosphoprotein implicated in the development of tubulointerstitial inflammation in various models of renal disease, in human diabetic kidneys, and to study the regulation of OPN expression in primary cultures of human renal proximal tubular epithelial cells (RPTEC). METHODS AND RESULTS: Differential gene expression profiling through subtractive hybridization demonstrated increased renal OPN mRNA expression in a patient with diabetic nephropathy. Immunohistochemical staining of normal and diabetic human kidney samples confirmed that OPN was localized to cortical tubular, interstitial and juxtaglomerular compartments. Quantification of OPN immunostaining revealed a marked increase in the percentage of OPN-positive tubular profiles in diabetic kidneys (47 +/- 9% versus 5 +/- 3%, diabetic versus minimal change disease) that correlated strongly with the degree of cortical scarring (r2 = 0.91). Results of Northern hybridization, flow cytometry and Western blotting indicated that glucose up-regulates OPN mRNA and protein expression in primary cultures of human RPTECs. This effect was independent of the osmotic effects of glucose and independent of insulin. Finally, glucose-stimulated OPN expression was inhibited by LY294002, an inhibitor of phosphatidylinositol 3-kinase activity, in a dose-dependent manner. CONCLUSIONS: OPN is expressed in human diabetic kidneys and regulation of OPN expression is via a glucose-mediated, phosphatidylinositol 3-kinase-dependent pathway.
Subject(s)
Diabetic Nephropathies/complications , Fibrosis/metabolism , Glucose/pharmacology , Kidney Tubules, Proximal/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Sialoglycoproteins/metabolism , Aged , Amino Acid Sequence , Blotting, Northern , Blotting, Western , Cells, Cultured , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/pathology , Enzyme Inhibitors/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/pathology , Fibrosis/etiology , Fibrosis/pathology , Flow Cytometry , Gene Expression Profiling , Humans , Immunohistochemistry , In Situ Hybridization , Kidney Tubules, Proximal/drug effects , Kidney Tubules, Proximal/pathology , Male , Molecular Sequence Data , Nephritis, Interstitial/metabolism , Nephritis, Interstitial/pathology , Osteopontin , Phosphatidylinositol 3-Kinases/drug effects , RNA, Messenger/analysis , Sequence Homology, Amino Acid , Sialoglycoproteins/drug effectsABSTRACT
Ribonucleotide reductase R1 gene expression is elevated in BALB/c 3T3 fibroblasts treated with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). We show that TPA treatment increased the half-life of R1 mRNA by 5-fold, indicating that TPA regulates R1 gene expression by a post-transcriptional mechanism. We investigated the possibility that the 3' untranslated region (3'UTR) of R1 mRNA contains regulatory information for TPA-mediated message stability. Our studies demonstrated that a 49 nucleotide (nt) TPA-responsive region existed within the R1 mRNA 3'UTR. Deletion of the 49 nt region led to the abolishment of TPA-induced stability of R1 and hybrid CAT mRNAs. Further deletions of the 3'UTR did not significantly affect mRNA turnover rates. In addition, we detected by cross-linking a novel 52-57 kDa R1 mRNA-binding protein (R1BP) that bound selectively to the 49 nt region of the R1 mRNA 3'UTR and did not bind to the 5'UTR, the coding region or other mRNA 3'UTRs. The R1BP-RNA binding activity observed in unstimulated cells was rapidly and markedly down-regulated after TPA treatment, suggesting a role for R1BP in mRNA metabolism, and in the mechanism of action of TPA-induced R1 message stabilization. These results support a novel model of R1 gene regulation in which a cis-element(s) within the 49 nt region of the R1 mRNA 3'UTR interacts with R1BP in a mechanism that regulates R1 message stability. We propose that this model accounts for the TPA-mediated stability alteration of R1 message, through down-regulation of R1BP-RNA binding activity linked to a reduction in the rate of R1 mRNA degradation.
Subject(s)
Introns , RNA, Messenger/metabolism , Ribonucleotide Reductases/genetics , Tetradecanoylphorbol Acetate/pharmacology , 3T3 Cells , Animals , Base Sequence , Cell Line , Chloramphenicol O-Acetyltransferase/genetics , Cloning, Molecular , Cytosol/metabolism , Down-Regulation , Half-Life , Mice , Mice, Inbred BALB C , Molecular Sequence Data , RNA, Messenger/chemistry , RNA-Binding Proteins/metabolism , Restriction Mapping , TransfectionABSTRACT
Ribonucleotide reductase R2 gene expression is elevated in BALB/c 3T3 fibroblasts treated with transforming growth factor beta 1. We investigated the possibility that the 3'-UTR of ribonucleotide reductase R2 mRNA contains regulatory information for TGF-beta 1 induced message stability. Using end-labeled RNA fragments in gel shift assays and UV cross-linking analyses, we detected in the 3'-UTR a novel 9 nucleotide (nt) cis element, 5'-GAGUUUGAG-3' site, which interacted specifically with a cytosolic protease sensitive factor to form a 75 kDa complex. The cis element protein binding activity was inducible and markedly up-regulated cross-link 4 h after TGF-beta 1 treatment of mouse BALB/c 3T3 cells. Other 3'-UTRs [IRE, GM-CSF, c-myc and homopolymer (U)] were poor competitors to the cis element with regard to forming the TGF-beta 1 dependent RNA-protein complex. However, the cis element effectively competed out the formation of the R2 3'-UTR protein complex. Cytosolic extracts from a variety of mammalian cell lines (monkey Cos7, several mouse fibrosarcomas and human HeLa S3) demonstrated similar TGF-beta 1 dependent RNA-protein band shifts as cell extract from BALB/c 3T3 mouse fibroblasts. Binding was completely prevented by several different mutations within the cis element, and by substitution mutagenesis, we were able to predict the consensus sequences, 5'-GAGUUUNNN-3' and 5'-NNNUUUGAG-3' for optimal protein binding. These results support a model in which the 9 nt region functions in cis to destabilize R2 mRNA in cells; and upon activation, a TGF-beta 1 responsive protein is induced and interacts with the 9 nt cis element in a mechanism that leads to stabilization of the mRNA. This appears to be the first example of a mRNA binding site that is involved in TGF-beta 1-mediated effects.
Subject(s)
RNA, Messenger/genetics , Ribonucleotide Reductases/genetics , Transforming Growth Factor beta/metabolism , 3T3 Cells , Animals , Base Sequence , Binding Sites/genetics , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Mutation , Protein Binding/genetics , RNA, Messenger/metabolism , Sequence Analysis , Transforming Growth Factor beta/pharmacologyABSTRACT
A rate-limiting reaction in DNA synthesis is catalyzed by ribonucleotide reductase, the enzyme responsible for reducing ribonucleotides to provide the deoxyribonucleotide precursors of DNA. In this study, we have tested the hypothesis that posttranscriptional regulation of ribonucleotide reductase R1 gene expression is controlled by a protein kinase C signal pathway. We show that mouse BALB/c 3T3 fibroblasts treated with the potent and highly specific protein kinase C inhibitor bisindolylmaleimide GF 109203X contain significantly reduced steady-state levels of R1 mRNA and protein. Message half-life studies demonstrate that this is due, at least in part, to a marked decrease in R1 message stability in cells treated with the protein kinase C inhibitor. Furthermore, the protein kinase C signal pathway appears to be specifically involved in this regulation since 8-bromo-cAMP, a modulator of the protein kinase A pathway, had no effect on R1 mRNA levels or stability properties. Cross-linking assays revealed that the binding activity of a R1 mRNA 3'-untranslated region binding protein (R1BP), which was previously shown to be involved in the regulation of R1 mRNA stability, was significantly elevated after treatment of the cells with GF 109203X, in a dose-dependent manner. However, treatment with 8-bromo-cAMP at concentrations up to 2.5 mM did not obviously affect the basic level of the R1BP-RNA interaction. These observations provide a better understanding of the biochemical signals that are critical for the cis-trans interaction-mediated posttranscriptional regulation of ribonucleotide reductase R1 gene expression.
Subject(s)
Gene Expression Regulation, Enzymologic , Protein Kinase C/metabolism , RNA, Messenger/metabolism , Ribonucleotide Reductases/genetics , Signal Transduction , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Animals , Cells, Cultured , Gene Expression Regulation, Enzymologic/drug effects , Indoles/pharmacology , Maleimides/pharmacology , Mice , Mice, Inbred BALB C , RNA Processing, Post-Transcriptional/drug effects , RNA-Binding Proteins/metabolism , Ribonucleotide Reductases/biosynthesisABSTRACT
Ribonucleotide reductase catalyses the reaction that eventually provides the four deoxyribonucleotides required for the synthesis and repair of DNA. U.v.-cross-linking and band-shift experiments have identified in COS 7 monkey cells an approx. 57 kDa ribonucleotide reductase R1 mRNA-binding protein called R1BP, which binds specifically to a 49-nt region of the R1 mRNA 3'-untranslated region (3'UTR). The R1BP-RNA binding activity was down-regulated by the tumour promoters phorbol 12-myristate 13-acetate (PMA; 'TPA') and okadaic acid, and up-regulated by the protein kinase C inhibitor staurosporine, in a dose-dependent fashion. Furthermore, staurosporine treatment decreased the stability of R1 and CAT (chloramphenicol acetyltransferase)/R1 hybrid mRNAs, whereas PMA and okadaic acid increased the stability of these messages, in a dose-dependent manner. In contrast, treatment of cells with forskolin, a protein kinase A inhibitor, did not alter either R1BP-RNA binding or R1 mRNA-stability characteristics. Transfectants containing R1 or CAT/R1 cDNA constructs with a deletion of the 49-nt 3'UTR sequence failed to respond in message-stability studies to the effects of PMA, staurosporine or okadaic acid. These observations indicate that a protein kinase C signal pathway regulates ribonucleotide reductase R1 gene expression post-transcriptionally, through a mechanism involving a specific cis-trans interaction at a 49-nt region within the R1 mRNA 3'UTR.
Subject(s)
Protein Kinase C/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Ribonucleotide Reductases/genetics , 3T3 Cells , Alkaloids/pharmacology , Animals , Binding Sites , Cell Line , Chloramphenicol O-Acetyltransferase/genetics , Ethers, Cyclic/pharmacology , Haplorhini , Mice , Mice, Inbred BALB C , Okadaic Acid , Protein Biosynthesis , Protein Kinase C/antagonists & inhibitors , RNA, Messenger/genetics , Ribonucleotide Reductases/metabolism , Signal Transduction , StaurosporineABSTRACT
The R2 gene of ribonucleotide reductase is elevated in BALB/c 3T3 fibroblasts treated with the tumor promotor, 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA treatment increased the half-life of the R2 message by 3-fold, showing that TPA regulates R2 gene expression by a post-transcriptional mechanism(s). A 20-nucleotide (nt) TPA-responsive region was found within the R2 mRNA 3'-untranslated region (3'UTR). Ultraviolet cross-linking detected a novel 45-kDa protein-R2 mRNA complex migration band that bound selectively to the 20-nt fragment and did not bind to the 5'UTR or the coding region of the R2 message, or to the 3'UTRs of mRNA from several other genes, or to the homopolymer poly(A) sequence. The-45 kDa protein-R2 mRNA binding activity observed in unstimulated cells was markedly down-regulated after TPA treatment. Deletion of a 201-nt region, containing the 20-nt sequence, from the 3'UTR caused stabilization of hybrid chloramphenicol acetyltransferase mRNA in the absence of TPA treatment. Furthermore, in vitro decay reaction mixtures supplemented with the 20-nt sense RNA transcript resulted in stabilization of R2 message. A model is presented of R2 message regulation in which a cis-element within the 20-nt sequence of the 3'UTR interacts with a cytosolic protein to form a 45-kDa protein-mRNA binding complex. The TPA-induced alteration of R2 message stability is at least in part due to the down-regulation of the 45-kDa protein-mRNA binding activity which is linked to a reduction in the rate of R2 mRNA degradation.
Subject(s)
Gene Expression Regulation, Enzymologic/drug effects , RNA Processing, Post-Transcriptional , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Ribonucleotide Reductases/biosynthesis , Tetradecanoylphorbol Acetate/pharmacology , 3T3 Cells , Animals , Base Sequence , Cytoplasm/metabolism , DNA, Complementary/metabolism , Kinetics , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Molecular Weight , Protein Binding , Protein Biosynthesis , RNA Probes , RNA-Binding Proteins/isolation & purification , Restriction MappingABSTRACT
Ribonucleotide reductase is a highly regulated rate-limiting enzyme activity in DNA synthesis, responsible for reducing ribonucleotides to their deoxyribonucleotide forms. Using 3'-end labeled RNA and band-shift and UV cross-linking analyses, we have identified a cis-element, 5'-CAAACUUC-3', within the 3'-untranslated region of the mammalian ribonucleotide reductase R1 mRNA, which binds a cytoplasmic protein in BALB/c 3T3 mouse cells, to form a 57 kDa RNA-protein complex. Sequence-specific binding was observed, and binding was prevented by several different mutations within the cis-element. We suggest that this cis-trans interaction plays a role in R1 mRNA stability.
Subject(s)
Cytoplasm/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Ribonucleotide Reductases/genetics , 3T3 Cells , Animals , Base Sequence , Binding Sites , Binding, Competitive , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Mice , Molecular Sequence Data , Mutation/physiology , RNA Probes/genetics , RNA Probes/metabolismABSTRACT
Mammalian ribonucleotide reductase is a highly regulated activity essential for DNA synthesis and repair. The 3'-untranslated region (3'-UTR) of mammalian ribonucleotide reductase R2 mRNA has been implicated in the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate-mediated stabilization of mouse BALB/c 3T3 R2 message. We investigated the possibility that the 3'-UTR contains regulatory information for R2 mRNA turnover. Using 3'-end-labeled RNA in gel shift and UV cross-linking analyses, we detected in the 3'-UTR a novel 9-nucleotide cis-element, 5'-UCGUGUGCU-3', which interacted with a widely distributed cellular cytosolic protease-sensitive factor(s) in a sequence-specific manner to form a 45-kDa R2 binding protein complex. The binding activity was redox-sensitive and down-regulated by 12-O-tetradecanoylphorbol-13-acetate and okadaic acid in a dose-dependent manner. Insertion of a 154-base pair fragment containing the cis-element led to markedly reduced accumulation of chloramphenicol acetyltransferase hybrid mRNA relative to the same insert carrying a series of G --> A mutations within this element that eliminated binding. We suggest that the 9-nucleotide region functions as a destabilizing element. These results provide a model for ribonucleotide reductase gene expression through a novel and specific mRNA cis-trans-interaction involving a phosphorylation signal pathway that leads to changes in the stability of R2 message.
Subject(s)
RNA, Messenger/metabolism , Regulatory Sequences, Nucleic Acid , Ribonucleotide Reductases/genetics , 3T3 Cells , Animals , Base Sequence , Cell Line , DNA Primers/chemistry , Gene Expression Regulation, Enzymologic , Genes , Humans , Hydrogen Bonding , Mice , Molecular Sequence Data , Nucleic Acid Conformation , Oxidation-Reduction , Phosphoproteins/metabolism , Protein Binding , RNA-Binding Proteins/metabolismABSTRACT
Ribonucleotide reductase is a highly regulated enzyme that provides the four deoxyribonucleotides required for DNA synthesis. Our studies showed that TGF-beta 1 treatment of BALB/c 3T3 mouse fibroblasts markedly elevated ribonucleotide reductase R2 mRNA levels, and also increased the half-life of R2 message by 4-fold from 1.5 h in untreated cells to 6 h in treated cells. We describe a novel 75 Kd sequence-specific cytoplasmic factor (p75) that binds selectively to a 83-nucleotide 3'-untranslated region of R2 mRNA and did not bind to the 5'UTR, the coding region of the R2 message or to the 3'UTRs of other mRNAs (from c-myc, GM-CSF and the iron responsive element from the transferrin receptor mRNA), or to the homopolymer poly(A) sequence. p75-RNA binding activity, which requires new protein synthesis, is not present in untreated cells, but is induced following TGF-beta 1 stimulation. The in vivo kinetics of appearance of p75 binding activity paralleled the accumulation of R2 mRNA. Insertion of the 3'-untranslated region into the chloramphenicol acetyltransferase (CAT) message confers TGF-beta 1 induced stability of RNA in stably transfected cells, while the same insert carrying a deletion of the 83-nucleotide fragment had little affect on RNA levels. Furthermore, in vitro decay reactions that contained the 83-nucleotide RNA or deletion of this fragment caused a significant decrease in TGF-beta 1 stabilization of R2 message. A model is presented of R2 message regulation in which TGF-beta 1 mediated stabilization of R2 message involves a specific interaction of a p75-trans-acting factor with a cis-element(s) stability determinant within the 83-nucleotide sequence which is linked to a reduction in the rate of R2 mRNA degradation.
Subject(s)
RNA, Messenger/metabolism , Ribonucleotide Reductases/genetics , Trans-Activators/metabolism , Transforming Growth Factor beta/metabolism , 3T3 Cells , Animals , Base Sequence , Binding, Competitive , Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol O-Acetyltransferase/metabolism , Cloning, Molecular , Cytoplasm/metabolism , Gene Expression Regulation , Introns , Kinetics , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Restriction Mapping , Ribonucleotide Reductases/metabolismABSTRACT
The receptor for hyaluronan mediated motility (RHAMM) gene expression is markedly elevated in fibrosarcomas exposed to transforming growth factor-beta1 (TGF-beta1). The half-life of RHAMM mRNA was increased by 3 fold in cells treated with TGF-beta1, indicating that growth factor regulation of RHAMM gene expression at least in part involves a posttranscriptional mechanism. Our studies demonstrated that a unique 30-nucleotide (nt) region that has three copies of the sequence, GCUUGC, was the TGF-beta1-responsive region in the 3'-untranslated region (3'-UTR) that mediated message stability. This region interacted specifically with cytoplasmic trans-factors to form multiple protein complexes of approximately 175, 97, 63, 26, and 17 kDa post-TGF-beta1 treatment, suggesting a role for these complexes in the mechanism of action of TGF-beta1-induced message stabilization. Insertion of the 3'-UTR into the chloramphenicol acetyltransferase gene conferred TGF-beta1 induced stability of chloramphenicol acetyltransferase-hybrid RNA in stably transfected cells, while the same insert carrying a deletion containing the 30-nt region had no significant effect on mRNA stability. These results provide a model of RHAMM message regulation in which TGF-beta1-mediated alteration of RHAMM message stability involves the up-regulation of multiple protein interactions with a 30-nt cis-element stability determinant in the 3'-UTR. This model also suggests that this 30-nt base region functions in cis to destabilize RHAMM mRNA in resting normal cells.
Subject(s)
Extracellular Matrix Proteins/biosynthesis , Gene Expression Regulation/drug effects , Hyaluronan Receptors/biosynthesis , RNA, Messenger/metabolism , Transforming Growth Factor beta/pharmacology , Animals , Base Sequence , Binding Sites , Cell Line , Chloramphenicol O-Acetyltransferase/biosynthesis , Fibrosarcoma , Mice , Molecular Sequence Data , Oligonucleotide Probes , Oligoribonucleotides , Protein Biosynthesis/drug effects , RNA, Messenger/chemistry , Recombinant Proteins/biosynthesis , Transcription, Genetic , Transfection , Tumor Cells, CulturedABSTRACT
The R2 component of ribonucleotide reductase is rate-limiting for DNA synthesis in proliferating cells, and recently, it has been shown that aberrant expression of R2 directly alters the malignant potential of tumor cells. We show that R2 gene expression is elevated in BALB/c 3T3 cells treated with transforming growth factor (TGF)-beta 1, TGF-beta 2, or TGF-beta 3, as determined by Northern blot analysis. Gel shift assays and UV crosslinking studies demonstrated similar post-transcriptional regulation at the 3'-untranslated region (3'-UTR) of the R2 mRNA, by TGF-beta 1, TGF-beta 2, and TGF-beta 3. The three growth factors induced a common 75 kDa RNA-protein complex. A 9 nucleotide sequence, GAGUUUGAG, previously shown to be responsive to TGF-beta 1-mediated R2 message stability changes, effectively competed out the formation of the R2 3'-UTR complex. We propose that these three different members of the TGF-beta family work through a common mechanism to control an important component of cell proliferation and a potential determinant of malignant progression.