scholarly journals Control of c-myc mRNA half-life in vitro by a protein capable of binding to a coding region stability determinant.

1992 ◽  
Vol 6 (4) ◽  
pp. 642-654 ◽  
Author(s):  
P L Bernstein ◽  
D J Herrick ◽  
R D Prokipcak ◽  
J Ross
Keyword(s):  
Half Life ◽  
Coding Region ◽  
Stability Determinant ◽  
Mrna Half Life

scholarly journals Human La protein: a stabilizer of histone mRNA.

1997 ◽  
Vol 17 (6) ◽  
pp. 3028-3036 ◽  
Author(s):  
R S McLaren ◽  
N Caruccio ◽  
J Ross
Keyword(s):  
Half Life ◽  
Mrna Decay ◽  
Rna Binding ◽  
S Phase ◽  
Coding Region ◽  
Histone Mrna ◽  
Histone Proteins ◽  
Unbound Fraction ◽  
Mrna Half Life

Histone mRNA is destabilized at the end of S phase and in cell-free mRNA decay reaction mixtures supplemented with histone proteins, indicating that histones might autoregulate the histone mRNA half-life. Histone mRNA destabilization in vitro requires three components: polysomes, histones, and postpolysomal supernatant (S130). Polysomes are the source of the mRNA and mRNA-degrading enzymes. To investigate the role of the S130 in autoregulation, crude S130 was fractionated by histone-agarose affinity chromatography. Two separate activities affecting the histone mRNA half-life were detected. The histone-agarose-bound fraction contained a histone mRNA destabilizer that was activated by histone proteins; the unbound fraction contained a histone mRNA stabilizer. Further chromatographic fractionation of unbound material revealed only a single protein stabilizer, which was purified to homogeneity, partially sequenced, and found to be La, a well-characterized RNA-binding protein. When purified La was added to reaction mixtures containing polysomes, a histone mRNA decay intermediate was stabilized. This intermediate corresponded to histone mRNA lacking 12 nucleotides from its 3' end and containing an intact coding region. Anti-La antibody blocked the stabilization effect. La had little or no effect on several other cell cycle-regulated mRNAs. We suggest that La prolongs the histone mRNA half-life during S phase and thereby increases histone protein production.

Glucocorticoids regulate NHE-3 transcription in OKP cells

1996 ◽  
Vol 270 (1) ◽  
pp. F164-F169 ◽  
Author(s):  
M. Baum ◽  
M. Amemiya ◽  
V. Dwarakanath ◽  
R. J. Alpern ◽  
O. W. Moe
Keyword(s):  
Gene Transcription ◽  
Half Life ◽  
Time Course ◽  
In Vitro Transcription ◽  
Mrna Abundance ◽  
Proton Secretion ◽  
Threefold Increase ◽  
Mrna Half Life ◽  
Synthetic Glucocorticoid

OKP cells express NHE-3, an amiloride-resistant Na+/H+ antiporter, which is likely an isoform responsible for apical proton secretion by the proximal tubule. We have previously shown that an amiloride-resistant Na+/H+ antiporter in OKP cells is regulated by dexamethasone, a synthetic glucocorticoid. The purpose of the present study was to examine the mechanism for the glucocorticoid-mediated increase in Na+/H+ antiporter activity. Incubation of OKP cells with 10(-6) M dexamethasone resulted in a two- to threefold increase in NHE-3 mRNA abundance. This increase was seen after 4 h of incubation with dexamethasone, a time course similar to that found for Na+/H+ antiporter activity. To examine the mechanism for the increase in NHE-3 mRNA abundance, mRNA half-life and in vitro transcription experiments were performed. NHE-3 mRNA had a half-life of 8 h in control and dexamethasone-treated cells. The rate of in vitro transcription was 1.8-fold greater when OKP cells were treated with dexamethasone. These data suggest that the glucocorticoid-mediated increase in Na+/H+ antiporter activity is due to an increase in NHE-3 gene transcription.

A Dictyostelium prespore-specific gene is transcriptionally repressed by DIF in vitro

Development ◽  
1988 ◽  
Vol 103 (3) ◽  
pp. 519-524 ◽  
Author(s):  
A.E. Early ◽  
J.G. Williams
Keyword(s):  
Half Life ◽  
Stalk Cell ◽  
Specific Gene ◽  
Transcriptional Level ◽  
Mrna Half Life ◽  
Spore Cell ◽  
Transcriptional Controls

One important role of DIF, the stalk cell-specific inducer of Dictyostelium, may be to divert cells from the spore cell pathway of differentiation. The D19 gene encodes an mRNA which is highly enriched in prespore over prestalk cells in the migratory slug. We show, using a mutant defective in DIF accumulation, that the concentration of D19, and several other prespore mRNA sequences, decreases in the presence of exogenous DIF. There is evidence that both transcriptional and post-transcriptional controls operate to regulate expression of these genes. We have performed in vitro nuclear transcription and mRNA half-life analyses, and find that DIF acts at the transcriptional level to repress the accumulation of the D19 mRNA.

scholarly journals Mechanism for Stabilizing mRNAs Involved in Methanol-Dependent Methanogenesis of Cold-Adaptive Methanosarcina mazei zm-15

2013 ◽  
Vol 80 (4) ◽  
pp. 1291-1298 ◽  
Author(s):  
Yi Cao ◽  
Jie Li ◽  
Na Jiang ◽  
Xiuzhu Dong
Keyword(s):  
Half Life ◽  
Content Type ◽  
Methanosarcina Mazei ◽  
Reverse Transcription Quantitative Pcr ◽  
Aceticlastic Methanogenesis ◽  
The Stability ◽  
Zoige Wetland ◽  
Mrna Half Life

ABSTRACTMethylotrophic methanogenesis predominates at low temperatures in the cold Zoige wetland in Tibet. To elucidate the basis of cold-adapted methanogenesis in these habitats,Methanosarcina mazeizm-15 was isolated, and the molecular basis of its cold activity was studied. For this strain, aceticlastic methanogenesis was reduced 7.7-fold during growth at 15°C versus 30°C. Methanol-derived methanogenesis decreased only 3-fold under the same conditions, suggesting that it is more cold adaptive. Reverse transcription-quantitative PCR (RT-qPCR) detected <2-fold difference in the transcript abundances ofmtaA1,mtaB1, andmtaC1, the methanol methyltransferase (Mta) genes, in 30°C versus 15°C culture, whileackAandptamRNAs, encoding acetate kinase (Ack) and phosphotransacetylase (Pta) in aceticlastic methanogenesis, were 4.5- and 6.8-fold higher in 30°C culture than in 15°C culture. Thein vivohalf-lives ofmtaA1andmtaC1B1mRNAs were similar in 30°C and 15°C cultures. However, thepta-ackAmRNA half-life was significantly reduced in 15°C culture compared to 30°C culture. Using circularized RNA RT-PCR, large 5′ untranslated regions (UTRs) (270 nucleotides [nt] and 238 nt) were identified formtaA1andmtaC1B1mRNAs, while only a 27-nt 5′ UTR was present in thepta-ackAtranscript. Removal of the 5′ UTRs significantly reduced thein vitrohalf-lives ofmtaA1andmtaC1B1mRNAs. Remarkably, fusion of themtaA1ormtaC1B15′ UTRs topta-ackAmRNA increased itsin vitrohalf-life at both 30°C and 15°C. These results demonstrate that the large 5′ UTRs significantly enhance the stability of the mRNAs involved in methanol-derived methanogenesis in the cold-adaptiveM. mazeizm-15.

scholarly journals A point mutation in the coding region of uroporphyrinogen decarboxylase associated with familial porphyria cutanea tarda

Blood ◽  
1989 ◽  
Vol 73 (4) ◽  
pp. 892-895
Author(s):  
JR Garey ◽  
JL Hansen ◽  
LM Harrison ◽  
JB Kennedy ◽  
JP Kushner
Keyword(s):  
Point Mutation ◽  
Half Life ◽  
Porphyria Cutanea Tarda ◽  
Amino Acid Position ◽  
Mutant Protein ◽  
Mrna Levels ◽  
Coding Region ◽  
Uroporphyrinogen Decarboxylase

Familial porphyria cutanea tarda (PCT) is inherited as an autosomal dominant trait caused by decreased activity of uroporphyrinogen decarboxylase (URO-D). In most families with PCT, URO-D mRNA levels are normal but both catalytic activity and immunologic reactivity of URO-D are half normal. We have cloned and sequenced 8 URO-D cDNA transcripts derived from a pedigree member with familial PCT. Three of the cDNAs had sequences encoding normal URO-D but five cDNA's contained a point mutation resulting in a gly----val substitution at amino acid position 281. An oligonucleotide probe complementary to the mutant sequence hybridized to DNA from affected individuals within the pedigree, but not to DNA from normal individuals. Measurements of pulse labeled URO-D in Epstein-Barr virus transformed lymphocytes indicated that the mutant protein has a half-life in vivo of less than four hours. In vitro measurements utilizing labeled URO-Ds generated in a reticulocyte lysate system revealed a 12-hour half-life for the mutant protein compared with a 102-hour half-life for normal URO-D. This is the first URO-D mutation to be characterized in a pedigree with familial PCT. This mutation was not detected in affected individuals from seven other PCT pedigrees, suggesting that PCT can result from different mutations.

scholarly journals Hormonal regulation of cytochrome P450 aromatase mRNA stability in non-luteinizing bovine granulosa cells in vitro

2006 ◽  
Vol 190 (1) ◽  
pp. 107-115 ◽  
Author(s):  
Malha Sahmi ◽  
Edmir S Nicola ◽  
Christopher A Price
Keyword(s):  
Cytochrome P450 ◽  
Granulosa Cells ◽  
Half Life ◽  
Hormonal Regulation ◽  
Hormonal Control ◽  
Transcriptional Level ◽  
P450 Aromatase ◽  
Cytochrome P450 Aromatase ◽  
Mrna Half Life

In the present study, we determined the potential for post-transcriptional regulation of cytochrome P450 aromatase (Cyp19), cytochrome P450 side-chain cleavage (Cyp11a) and 17β-hydroxysteroid dehydrogenase I (Hsd17b1) mRNA. Bovine granulosa cells were cultured in non-luteinizing conditions that permit long-term oestradiol secretion. Half-lives of mRNA were measured by Northern and/or reverse transcriptase (RT)-PCR after inhibition of gene transcription. In FSH-stimulated cells, the Cyp11a and Hsd17b1 mRNAs had half-lives greater than 12 h. The half-life of Cyp19 mRNA was significantly shorter at 3 h. The addition of the translation inhibitor cycloheximide to FSH-stimulated cells significantly increased Cyp19 mRNA half-life to approximately 12 h. Stimulation of cells with insulin resulted in Cyp19 mRNA half-life that was double (P<0.05) that in FSH-stimulated cells. We conclude that bovine Cyp19 mRNA is very labile under physiological conditions, and that Cyp19 expression is under hormonal control at a post-transcriptional level.

scholarly journals Premature translation termination mediates triosephosphate isomerase mRNA degradation.

1988 ◽  
Vol 8 (2) ◽  
pp. 802-813 ◽  
Author(s):  
I O Daar ◽  
L E Maquat
Keyword(s):  
Amino Acids ◽  
Mrna Stability ◽  
Half Life ◽  
Triosephosphate Isomerase ◽  
Stop Codon ◽  
Translation Termination ◽  
Mrna Structure ◽  
Mrna Half Life ◽  
Premature Translation Termination

We characterized an anemia-inducing mutation in the human gene for triosephosphate isomerase (TPI) that resulted in the production of prematurely terminated protein and mRNA with a reduced cytoplasmic half-life. The mutation converted a CGA arginine codon to a TGA nonsense codon and generated a protein of 188 amino acids, instead of the usual 248 amino acids. To determine how mRNA primary structure and translation influence mRNA stability, in vitro-mutagenized TPI alleles were introduced into cultured L cells and analyzed for their effect on TPI RNA metabolism. Results indicated that mRNA stability is decreased by all nonsense and frameshift mutations. To determine the relative contribution of the changes in mRNA structure and translation to the altered half-life, the effects of individual mutations were compared with the effects of second-site reversions that restored translation termination to normal. All mutations that resulted in premature translation termination reduced the mRNA half-life solely or mainly by altering the length of the mRNA that was translated. The only mutation that altered translation termination and that reduced the mRNA half-life mainly by affecting the mRNA structure was an insertion that shifted termination to a position downstream of the normal stop codon.

Premature translation termination mediates triosephosphate isomerase mRNA degradation

1988 ◽  
Vol 8 (2) ◽  
pp. 802-813 ◽  
Author(s):  
I O Daar ◽  
L E Maquat
Keyword(s):  
Amino Acids ◽  
Mrna Stability ◽  
Half Life ◽  
Triosephosphate Isomerase ◽  
Stop Codon ◽  
Translation Termination ◽  
Mrna Structure ◽  
Mrna Half Life ◽  
Premature Translation Termination

We characterized an anemia-inducing mutation in the human gene for triosephosphate isomerase (TPI) that resulted in the production of prematurely terminated protein and mRNA with a reduced cytoplasmic half-life. The mutation converted a CGA arginine codon to a TGA nonsense codon and generated a protein of 188 amino acids, instead of the usual 248 amino acids. To determine how mRNA primary structure and translation influence mRNA stability, in vitro-mutagenized TPI alleles were introduced into cultured L cells and analyzed for their effect on TPI RNA metabolism. Results indicated that mRNA stability is decreased by all nonsense and frameshift mutations. To determine the relative contribution of the changes in mRNA structure and translation to the altered half-life, the effects of individual mutations were compared with the effects of second-site reversions that restored translation termination to normal. All mutations that resulted in premature translation termination reduced the mRNA half-life solely or mainly by altering the length of the mRNA that was translated. The only mutation that altered translation termination and that reduced the mRNA half-life mainly by affecting the mRNA structure was an insertion that shifted termination to a position downstream of the normal stop codon.

O2 regulates surfactant protein A mRNA transcription and stability in human fetal lung in vitro

1998 ◽  
Vol 274 (3) ◽  
pp. L343-L350 ◽  
Author(s):  
Michael J. Acarregui ◽  
Ashish R. Kumar ◽  
Scott T. Penisten ◽  
Jeanne M. Snyder
Keyword(s):  
Half Life ◽  
Protein A ◽  
Fetal Lung ◽  
Surfactant Protein ◽  
Mrna Levels ◽  
Mrna Transcription ◽  
Preferential Expression ◽  
Mrna Half Life ◽  
Human Fetal Lung

The effect of O2 on surfactant protein (SP) A mRNA transcription and half-life was determined in midtrimester human fetal lung tissue cultured in either 20 (control) or 70% O2. Incubation of tissues in 70% O2 resulted in a 133% increase in SP-A mRNA transcription rate compared with control tissues. The SP-A mRNA half-life was increased by 54% in lung tissues cultured in 70% O2 vs. control tissues. Western blot analysis indicated a threefold increase in SP-A in the 70% O2 condition, demonstrating that O2 regulation of SP-A mRNA levels results in corresponding changes in SP-A levels. Primer extension assays were performed to determine whether the observed increase in SP-A mRNA levels is secondary to the preferential expression of one of the human SP-A genes, SP-A1 or SP-A2. Transcripts of both the SP-A1 and SP-A2 genes were increased ∼100% in tissues maintained in 70% O2 compared with control tissues. These data demonstrate that O2regulates human SP-A mRNA levels by both transcriptional and posttranscriptional mechanisms. Furthermore, because there is no differential effect of O2 on the expression of SP-A1 vs. SP-A2 mRNA, the properties of these genes that mediate regulation by O2 must be conserved between the two genes.

scholarly journals A point mutation in the coding region of uroporphyrinogen decarboxylase associated with familial porphyria cutanea tarda

Blood ◽  
1989 ◽  
Vol 73 (4) ◽  
pp. 892-895 ◽  
Author(s):  
JR Garey ◽  
JL Hansen ◽  
LM Harrison ◽  
JB Kennedy ◽  
JP Kushner
Keyword(s):  
Point Mutation ◽  
Half Life ◽  
Porphyria Cutanea Tarda ◽  
Amino Acid Position ◽  
Mutant Protein ◽  
Mrna Levels ◽  
Coding Region ◽  
Uroporphyrinogen Decarboxylase

Abstract Familial porphyria cutanea tarda (PCT) is inherited as an autosomal dominant trait caused by decreased activity of uroporphyrinogen decarboxylase (URO-D). In most families with PCT, URO-D mRNA levels are normal but both catalytic activity and immunologic reactivity of URO-D are half normal. We have cloned and sequenced 8 URO-D cDNA transcripts derived from a pedigree member with familial PCT. Three of the cDNAs had sequences encoding normal URO-D but five cDNA's contained a point mutation resulting in a gly----val substitution at amino acid position 281. An oligonucleotide probe complementary to the mutant sequence hybridized to DNA from affected individuals within the pedigree, but not to DNA from normal individuals. Measurements of pulse labeled URO-D in Epstein-Barr virus transformed lymphocytes indicated that the mutant protein has a half-life in vivo of less than four hours. In vitro measurements utilizing labeled URO-Ds generated in a reticulocyte lysate system revealed a 12-hour half-life for the mutant protein compared with a 102-hour half-life for normal URO-D. This is the first URO-D mutation to be characterized in a pedigree with familial PCT. This mutation was not detected in affected individuals from seven other PCT pedigrees, suggesting that PCT can result from different mutations.

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