scholarly journals Role of the 5′-untranslated region of mRNA in the synthesis of S-adenosylmethionine decarboxylase and its regulation by spermine

1994 ◽  
Vol 302 (3) ◽  
pp. 765-772 ◽  
Author(s):  
L M Shantz ◽  
R Viswanath ◽  
A E Pegg
Keyword(s):  
Secondary Structure ◽  
Untranslated Region ◽  
Fold Increase ◽  
Negative Regulator ◽  
Mrna Levels ◽  
Coding Region ◽  
Polyamine Biosynthesis ◽  
Adenosylmethionine Decarboxylase ◽  
Spermine Synthase ◽  
Synthase Inhibitor

S-Adenosylmethionine decarboxylase (AdoMetDC), a rate-limiting enzyme in polyamine biosynthesis, is regulated by polyamines at the levels of both transcription and translation. Two unusual features of AdoMetDC mRNA are a long (320 nt) 5′-untranslated region (5′UTR), which is thought to contain extensive secondary structure, and a short (15 nt) open reading frame (ORF) within the 5′UTR. We have studied the effects of altering these elements on both the expression of AdoMetDC and its regulation by n-butyl-1,3-diaminopropane (BDAP), a spermine synthase inhibitor. Human AdoMetDC cDNAs containing alterations in the 5′UTR, as well as chimaeric constructs in which the AdoMetDC 5′UTR was inserted ahead of the luciferase-coding region, were transfected into COS-7 cells. Construct pSAM320, which contains all of the 5′UTR, the AdoMetDC protein-coding region and the 3′UTR, was expressed poorly (2-fold over the endogenous activity). Deletion of virtually the entire 5′UTR, leaving nt -12 to -1, increased expression 59-fold, suggesting that 5′UTR acts as a negative regulator. The same effect was seen when the 27 nt at the extreme 5′ end were removed (pSAM293, 47-fold increase), or when the internal ORF which is present in this region was destroyed by changing the ATG to CGA (pSAM320-ATG, 38-fold increase). The expression and regulation of pSAM44 (made by deleting nt -288 to -12), which has very little predicted secondary strucutre, was very similar to that of pSAM320 indicating that the terminal 27 nt including the internal ORF rather than extensive secondary structure may be responsible for the low basal levels of AdoMetDC expression. These results, confirmed using luciferase constructs, suggest that the negative effect on expression is predominantly due to the internal ORF. Depletion of spermine by BDAP increased the expression from pSAM320 more than 5-fold without affecting AdoMetDC mRNA levels. Expression from pSAM293 was unchanged by spermine depletion, whereas that from pSAM320-ATG was increased 2.5-fold. These results indicate the presence of a spermine response element in the first 27 nt of the 5′UTR that may include but is not entirely due to the internal ORF.

A preprogalanin cDNA from the turtle pituitary and regulation of its gene expression

2007 ◽  
Vol 292 (4) ◽  
pp. R1649-R1656 ◽  
Author(s):  
John Yuh-Lin Yu ◽  
Chin-Hon Pon ◽  
Hui-Chen Ku ◽  
Chih-Ting Wang ◽  
Yung-Hsi Kao
Keyword(s):  
Mrna Expression ◽  
Untranslated Region ◽  
Signal Sequence ◽  
Biological Effects ◽  
In Vitro Study ◽  
The Body ◽  
Mrna Levels ◽  
Coding Region ◽  
Reading Frame

Galanin is a hormone 29 or 30 amino acids (aa) long that is widely distributed within the body and exerts numerous biological effects in vertebrates. To fully understand its physiological roles in reptiles, we analyzed preprogalanin cDNA structure and expression in the turtle pituitary. Using the Chinese soft-shell turtle ( Pelodiscus sinensis order Testudines), we obtained a 672-base pair (bp) cDNA containing a 99-bp 5′-untranslated region, a 324-bp preprogalanin coding region, and a 249-bp 3′-untranslated region. The open-reading frame encoded a 108-aa preprogalanin protein with a putative 23-aa signal sequence at the NH2 terminus. Based on the location of putative Lys-Arg dibasic cleavage sites and an amidation signal of Gly-Lys-Arg, we propose that turtle preprogalanin is processed to yield a 29-aa galanin peptide with Gly1 and Thr29 substitutions and a COOH-terminal amidation. Sequence comparison revealed that turtle preprogalanin and galanin-29 had 48–81% and 76–96% aa identities with those of other vertebrates, respectively, suggesting their conservative nature. Expression of the turtle galanin gene was detected in the pituitary, brain, hypothalamus, stomach, liver, pancreas, testes, ovaries, and intestines, but not in the adipose or muscle tissues, suggesting tissue-dependent differences. An in vitro study that used pituitary tissue culture indicated that treatment with 17β-estradiol, testosterone, or gonadotropin-releasing hormone resulted in increased galanin mRNA expression with dose- or time-dependent differences, whereas leptin and neuropeptide Y reduced galanin mRNA levels. These results suggest a hormone-dependent effect on hypophyseal galanin mRNA expression.

scholarly journals Regulation of protein synthesis by eIF4E phosphorylation in adult cardiocytes: the consequence of secondary structure in the 5′-untranslated region of mRNA

2004 ◽  
Vol 378 (1) ◽  
pp. 73-82 ◽  
Author(s):  
William J. TUXWORTH ◽  
Atif N. SAGHIR ◽  
Laura S. SPRUILL ◽  
Donald R. MENICK ◽  
Paul J. McDERMOTT
Keyword(s):  
Protein Synthesis ◽  
Secondary Structure ◽  
Untranslated Region ◽  
Primary Cultures ◽  
Initiation Factor ◽  
Luciferase Reporter ◽  
Translational Efficiency ◽  
Mrna Levels ◽  
Hypertrophic Growth ◽  
Reporter Mrna

In adult cardiocytes, eIF4E (eukaryotic initiation factor 4E) activity and protein synthesis are increased concomitantly in response to stimuli that induce hypertrophic growth. We tested the hypothesis that increases in eIF4E activity selectively improve the translational efficiency of mRNAs that have an excessive amount of secondary structure in the 5´-UTR (5´-untranslated region). The activity of eIF4E was modified in primary cultures of adult cardiocytes using adenoviral gene transfer to increase either the amount of eIF4E or the extent of endogenous eIF4E phosphorylation. Subsequently, the effects of eIF4E on translational efficiency were assayed following adenoviral-mediated expression of luciferase reporter mRNAs that were either ‘stronger’ (less structure in the 5´-UTR) or ‘weaker’ (more structure in the 5´-UTR) with respect to translational efficiency. The insertion of G+C-rich repeats into the 5´-UTR doubled the predicted amount of secondary structure and was sufficient to reduce translational efficiency of the reporter mRNA by 48±13%. Translational efficiency of the weaker reporter mRNA was not significantly improved by overexpression of wild-type eIF4E when compared with the stronger reporter mRNA. In contrast, overexpression of the eIF4E kinase Mnk1 [MAP (mitogen-activated protein) kinase signal-integrating kinase 1] was sufficient to increase the translational efficiency of either reporter mRNA, independent of the amount of secondary structure in their respective 5´-UTRs. The increases in translational efficiency produced by Mnk1 occurred in association with corresponding decreases in mRNA levels. These findings indicate that the positive effect of eIF4E phosphorylation on translational efficiency in adult cardiocytes is coupled with the stability of mRNA.

scholarly journals Homocysteine Down-regulates Cellular Glutathione Peroxidase (GPx1) by Decreasing Translation

2005 ◽  
Vol 280 (16) ◽  
pp. 15518-15525 ◽  
Author(s):  
Diane E. Handy ◽  
Yufeng Zhang ◽  
Joseph Loscalzo
Keyword(s):  
Glutathione Peroxidase ◽  
Untranslated Region ◽  
Stop Codon ◽  
Vascular Dysfunction ◽  
Lipid Peroxides ◽  
Mrna Levels ◽  
Coding Region ◽  
Sv40 Promoter ◽  
Secis Element

Hyperhomocysteinemia contributes to vascular dysfunction and an increase in the risk of cardiovascular disease. An elevated level of homocysteinein vivoand in cell culture systems results in a decrease in the activity of cellular glutathione peroxidase (GPx1), an intracellular antioxidant enzyme that reduces hydrogen peroxide and lipid peroxides. In this study, we show that homocysteine interferes with GPx1 protein expression without affecting transcript levels. Expression of the selenocysteine (SEC)-containing GPx1 protein requires special translational cofactors to “read-through” a UGA-stop codon that specifies SEC incorporation at the active site of the enzyme. These factors include a selenocysteine incorporation sequence (SECIS) in the 3′-untranslated region of the GPx1 mRNA and cofactors involved in the biosynthesis and translational insertion of SEC. To monitor SEC incorporation, we used a reporter gene system that has a UGA codon within the protein-coding region of the luciferase mRNA. Addition of either the GPx1 or GPx3 SECIS element in the 3′-untranslated region of the luciferase gene stimulated read-through by 6–11-fold in selenium-replete cells; absence of selenium prevented translation. To alter cellular homocysteine production, we used methionine in the presence of aminopterin, a folate antagonist, co-administered with hypoxanthine and thymidine (HAT/Met). This treatment increased homocysteine levels in the media by 30% (p< 0.01) and decreased GPx1 enzyme activity by 45% (p= 0.0028). HAT/Met treatment decreased selenium-mediated read-through significantly (p< 0.001) in luciferase constructs containing the GPx1 or GPx3 SECIS element; most importantly, the suppression of selenium-dependent read-through was similar whether an SV40 promoter or the GPx1 promoter was used to drive transcription of the SECIS-containing constructs. Furthermore, HAT/Met had no effect on steady-state GPx1 mRNA levels but decreased GPx1 protein levels, suggesting that this effect is not transcriptionally mediated. These data support the conclusion that homocysteine decreases GPx1 activity by altering the translational mechanism essential for the synthesis of this selenocysteine-containing protein.

Localization of cardiac (alpha)-myosin heavy chain mRNA is regulated by its 3′ untranslated region via mechanical activity and translational block

1997 ◽  
Vol 110 (23) ◽  
pp. 2969-2978 ◽  
Author(s):  
P. Goldspink ◽  
W. Sharp ◽  
B. Russell
Keyword(s):  
Myosin Heavy Chain ◽  
Cardiac Myocytes ◽  
Heavy Chain ◽  
Untranslated Region ◽  
Contractile Activity ◽  
Intracellular Localization ◽  
Mechanical Activity ◽  
Mrna Levels ◽  
Coding Region ◽  
Reporter Protein

We have altered the spontaneous contractile activity of neonatal cardiac myocytes in culture to investigate the re-lationship between mechanical forces, myofibril assembly, and the localization and translation of (alpha)-myosin heavy chain mRNA. Immunofluorescence and in situ hybridization techniques revealed that contracting myocytes display well aligned myofibrils and a diffuse distribution of (alpha)-myosin heavy chain mRNA. Inhibition of contractile activity with the calcium channel blocker verapamil (10 microM) resulted in myofibril disassembly and a perinuclear mRNA distribution within six hours. There was a significant decrease (P&lt;0. 05) of mRNA levels, 5 to 15 micron away from the nucleus following 6 hours of verapamil treatment compared with control cells. Inhibition of protein synthesis with cycloheximide (10 microM) also resulted in perinuclear mRNA localization despite having little effect on contractile activity or myofibril assembly. To determine if the 3′ untranslated region of (alpha)-myosin heavy chain mRNA was sufficient for localizing the entire message, a chimeric construct composed of beta-galactosidase coding region followed by (alpha)-myosin heavy chain 3′ untranslated region sequences was made as a reporter plasmid and transfected into cultured myocytes. A perinuclear accumulation of ss-galactosidase was exhibited in many of the contractile arrested cells (48.3+/−2.4%, n=7). In contrast, significantly fewer (P&lt;0.05) contracting control (29.1+/−3.3%, n=7) and strongly contracting, isoproterenol-treated cells (27.2+/−6.1%, n=3) exhibited a perinuclear localization of protein. The distribution of the reporter protein was not affected by the contractile state in cells transfected with a constitutively translated 3′UTR. We propose that mechanical activity of neonatal cardiac myocytes regulates the intracellular localization of alpha-myosin heavy chain mRNA via the 3′ untranslated region mediated by an initial block in translation.

Molecular cloning of metallothionein cDNA and analysis of metallothionein gene expression in winter flounder tissues

1989 ◽  
Vol 67 (10) ◽  
pp. 2520-2527 ◽  
Author(s):  
King Ming Chan ◽  
William S. Davidson ◽  
Choy L. Hew ◽  
Garth L. Fletcher
Keyword(s):  
Cdna Library ◽  
Untranslated Region ◽  
Cadmium Chloride ◽  
Gill Filament ◽  
Winter Flounder ◽  
Mrna Levels ◽  
Pseudopleuronectes Americanus ◽  
Coding Region ◽  
Liver And Kidney ◽  
Time Required

Investigations into the precise role played by metallothionein (MT) in heavy-metal metabolism have been hampered by difficulties in positively identifying and quantifying MT in fish tissues. This study describes the development of an antisense MT RNA (cRNA) probe that will enable MT mRNA levels to be measured with a high degree of specificity and precision. Cadmium chloride administration induces the producton of MT mRNA in the liver and kidney of winter flounder (Pseudopleuronectes americanus). Poly(A)+ RNA purified from liver samples of winter flounder after cadmium chloride injections was used to construct a cDNA library. Several recombinant clones made complementary to MT mRNA were selected from this cDNA library by an oligonucleotide derived from the N-terminal amino acid sequence of winter flounder metallothionein. Sequence analysis of two of the cDNA inserts gave the structure of the entire 3′ untranslated region, a coding region corresponding to winter flounder MT and 49 nucleotides of the 5′ untranslated region. One of the flounder MT cDNAs, pWFMTC4, was subcloned into a RNA probe plasmid and transcribed to produce antisense MT RNA (cRNA). The MT cRNA was then used to detect the induction of MT mRNA production in the liver of winter flounder, following the administration of Cu2+, Zn2+, Cd2+, Pb2+, and Hg2+. The time required for the induction of hepatic MT mRNA by a single injection of Cd2+ was approximately 96 h. Dexamethasone did not induce an increase of MT mRNA in any of the winter flounder tissues examined (liver, kidney, heart, brain, intestinal scrape, and gill filament), whereas Cd2+ induced MT mRNA in all of the tissues except brain, where the constitutive level of expression was high.

scholarly journals Role of propylamine transferases in hormone-induced stimulation of polyamine biosynthesis

1980 ◽  
Vol 192 (1) ◽  
pp. 59-63 ◽  
Author(s):  
Kirsti Käpyaho ◽  
Hannu Pösö ◽  
Juhani Jänne
Keyword(s):  
Seminal Vesicle ◽  
Ornithine Decarboxylase ◽  
Ovarian Tissue ◽  
Ventral Prostate ◽  
Decarboxylase Activity ◽  
Spermidine Synthase ◽  
Polyamine Biosynthesis ◽  
Adenosylmethionine Decarboxylase ◽  
Spermine Synthase ◽  
Stimulation Of

The effect of various hormones on the activities of the four enzymes engaged with the biosynthesis of the polyamines has been investigated in the rat. Human choriogonadotropin induced a dramatic, yet transient, stimulation of l-ornithine decarboxylase (EC 4.1.1.17) activity in rat ovary, with no or only marginal changes in the activities of S-adenosyl-l-methionine decarboxylase (EC 4.1.1.50), spermidine synthase (aminopropyltransferase; EC 2.5.1.16) or spermine synthase. A single injection of oestradiol into immature rats maximally induced uterine ornithine decarboxylase at 4h after the injection. This early stimulation of ornithine decarboxylase activity was accompanied by a distinct enhancement of adenosylmethionine decarboxylase activity and a decrease in the activities of spermidine synthase and spermine synthase. In the seminal vesicle of castrated rats, testosterone treatment elicited a striking and persistent stimulation of ornithine decarboxylase and adenosylmethionine decarboxylase activities. The activity of spermidine synthase likewise rapidly increased between the first and the second day after the commencement of the hormone treatment, whereas the activity of spermine synthase remained virtually unchanged during the whole period of observation. Testosterone-induced changes in polyamine formation in the ventral prostate were comparable with those found in the seminal vesicle, with the possible exception of a more pronounced stimulation of spermidine synthase activity. It thus appears that an enhancement in one or both of the propylamine transferase (aminopropyltransferase) activities in response to hormone administration is an indicator of hormone-dependent growth (uterus and the male accessory sexual glands), and is not necessarily associated with non-proliferative hormonal responses, such as gonadotropin-induced luteinization of the ovarian tissue.

3′-Untranslated region of the type 2 bradykinin receptor is a potent regulator of gene expression

2006 ◽  
Vol 290 (2) ◽  
pp. F456-F464 ◽  
Author(s):  
Rocio Zamorano ◽  
Sunil Suchindran ◽  
James V. Gainer
Keyword(s):  
Gene Expression ◽  
Untranslated Region ◽  
Tandem Repeats ◽  
Heterologous Gene Expression ◽  
Mrna Level ◽  
Human Kidney ◽  
Variable Number ◽  
Negative Regulator ◽  
Coding Region

Regulation of the constitutively expressed type 2 bradykinin (B2) receptor, which mediates the principal actions of bradykinin, occurs at multiple levels. The goal of the current study was to determine whether the human B2 3′-untranslated region (UTR) has effects on gene expression, with particular focus on the variable number of tandem repeats (B2-VNTR) polymorphic portion of the 3′-UTR and its flanking AU-rich elements (AREs). When inserted downstream of the luciferase coding region of the pGL3-Promoter vector, the B2-VNTR reduced reporter gene activity by 85% compared with pGL3-Promoter alone (promoter control; P < 0.001), an effect that was not appreciably affected by mutation of the flanking AREs. The negative regulatory effects of the B2-VNTR region were position and orientation dependent and strongly positively correlated with the number of tandem repeats in the B2-VNTR region ( r = 0.85, P < 0.001). With respect to mechanism, quantitative RT-PCR revealed that the B2-VNTR mRNA level was 32% of that of promoter control ( P = 0.008), whereas the number of polyadenylated transcripts was 4% ( P = 0.02). In contrast, the mRNA half-life of the B2-VNTR was increased (B2-VNTR: 14.9 vs. promoter control: 12.2 h, P = 0.009). Transient transfection of human kidney-derived tsA201 cells with the B2-VNTR construct increased transcription of the native B2 receptor mRNA by 43% ( P < 0.05), supporting an endogenous B2 receptor-regulatory capacity of the B2-VNTR. In conclusion, these results identify novel pretranslational effects of the B2-VNTR region to act as a potent negative regulator of heterologous gene expression and support the notion that the bradykinin B2 3′-UTR may impact endogenous receptor regulation.

scholarly journals Increase in S-adenosylmethionine decarboxylase in SV-3T3 cells treated with S-methyl-5′-methylthioadenosine

1987 ◽  
Vol 244 (1) ◽  
pp. 49-54 ◽  
Author(s):  
A E Pegg ◽  
R Wechter ◽  
A Pajunen
Keyword(s):  
Translation Efficiency ◽  
3T3 Cells ◽  
Enzyme Protein ◽  
Polyamine Synthesis ◽  
Adenosylmethionine Decarboxylase ◽  
Spermine Synthase ◽  
Additional Stabilization ◽  
Key Enzyme ◽  
Translatable Mrna ◽  
Synthase Inhibitor

Treatment of SV-3T3 cells with the spermine synthase inhibitor S-methyl-5′-methylthioadenosine [AdoS+(CH3)2] led to a large increase in the activity of S-adenosylmethionine decarboxylase (AdoMetDC) without affecting ornithine decarboxylase. The elevation of AdoMetDC activity was due to an increased amount of enzyme protein, as demonstrated by radioimmunoassay and by immunoblotting. The increase in AdoMetDC protein was caused by at least three factors: an increase in the amount of translatable mRNA, an increased translation efficiency of the mRNA and an increase in the half-life of the protein. The depletion of spermine brought about by AdoS+(CH3)2 appeared to be responsible for the increased synthesis of AdoMetDC and for part of the decrease in its rate of degradation. An additional stabilization of the enzyme protein was probably due to the binding of AdoS+(CH3)2, which is also a weak inhibitor of AdoMetDC. These results demonstrate the importance of cellular spermine concentrations in regulating the activity of AdoMetDC, which is a key enzyme controlling polyamine synthesis.

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