scholarly journals Intranuclear defect in beta-globin mRNA accumulation due to a premature translation termination codon

Blood ◽  
1984 ◽  
Vol 64 (1) ◽  
pp. 13-22
Author(s):  
K Takeshita ◽  
BG Forget ◽  
A Scarpa ◽  
EJ Jr Benz
Keyword(s):  
Translation Termination ◽  
Beta Globin ◽  
Mrna Synthesis ◽  
Globin Mrna ◽  
Mrna Accumulation ◽  
S1 Nuclease ◽  
Translation Termination Codon ◽  
Premature Translation Termination ◽  
Premature Translation Termination Codon

We have analyzed a cloned beta O-thalassemia (beta O-thal) gene from a patient doubly heterozygous for hemoglobin Lepore and beta O- thalassemia. Studies of 3H-uridine incorporation into beta-globin mRNA in this patient's erythroblasts suggested an intranuclear defect in both beta and Lepore (delta beta) mRNA synthesis, as did S1 nuclease analysis of nuclear RNA. However, the nucleotide sequence of the beta O- thal gene revealed only a single base change in codon 39 (CAG----UAG), which created a premature translation termination codon. The 5′ flanking sequence, including transcription promotor boxes and the mRNA initiation (CAP) site, were normal. The unexpected effect of this mutation on intranuclear beta-mRNA synthesis in vivo was studied by insertion of the cloned gene into a plasmid expression vector and transfection into tissue culture (COS-1) cells. beta-Globin mRNA produced by the transfected cells was assessed by S1 nuclease analysis. The beta O-39 thalassemia gene generated five- to tenfold less beta- mRNA than a normal beta-gene in both nuclear and cytoplasmic RNA, simulating the results observed in vivo. Moreover, the small amount of beta O-39 mRNA produced was as stable as normal beta-mRNA during an actinomycin D chase, ruling out rapid cytoplasmic turnover as a cause of the reduced accumulation. Cotransfection of the beta O-39 thalassemia gene with a mutant tyrosine suppressor tRNA gene resulted in restoration of the beta O-39 mRNA accumulation to near-normal levels. On the basis of these results, we suggest that the low levels of beta-mRNA known to exist in the common form of beta O-thalassemia, beta O-39 thalassemia, result from a lesion in transcription, or early posttranscriptional processes; the defect appears to be corrected by restoration of proper translational potential to the mutant mRNA, at least in a gene transfer-expression system in tissue-culture cells.

scholarly journals Intranuclear defect in beta-globin mRNA accumulation due to a premature translation termination codon

Blood ◽  
1984 ◽  
Vol 64 (1) ◽  
pp. 13-22 ◽  
Author(s):  
K Takeshita ◽  
BG Forget ◽  
A Scarpa ◽  
EJ Jr Benz
Keyword(s):  
Translation Termination ◽  
Beta Globin ◽  
Mrna Synthesis ◽  
Globin Mrna ◽  
Mrna Accumulation ◽  
S1 Nuclease ◽  
Translation Termination Codon ◽  
Premature Translation Termination ◽  
Premature Translation Termination Codon

Abstract We have analyzed a cloned beta O-thalassemia (beta O-thal) gene from a patient doubly heterozygous for hemoglobin Lepore and beta O- thalassemia. Studies of 3H-uridine incorporation into beta-globin mRNA in this patient's erythroblasts suggested an intranuclear defect in both beta and Lepore (delta beta) mRNA synthesis, as did S1 nuclease analysis of nuclear RNA. However, the nucleotide sequence of the beta O- thal gene revealed only a single base change in codon 39 (CAG----UAG), which created a premature translation termination codon. The 5′ flanking sequence, including transcription promotor boxes and the mRNA initiation (CAP) site, were normal. The unexpected effect of this mutation on intranuclear beta-mRNA synthesis in vivo was studied by insertion of the cloned gene into a plasmid expression vector and transfection into tissue culture (COS-1) cells. beta-Globin mRNA produced by the transfected cells was assessed by S1 nuclease analysis. The beta O-39 thalassemia gene generated five- to tenfold less beta- mRNA than a normal beta-gene in both nuclear and cytoplasmic RNA, simulating the results observed in vivo. Moreover, the small amount of beta O-39 mRNA produced was as stable as normal beta-mRNA during an actinomycin D chase, ruling out rapid cytoplasmic turnover as a cause of the reduced accumulation. Cotransfection of the beta O-39 thalassemia gene with a mutant tyrosine suppressor tRNA gene resulted in restoration of the beta O-39 mRNA accumulation to near-normal levels. On the basis of these results, we suggest that the low levels of beta-mRNA known to exist in the common form of beta O-thalassemia, beta O-39 thalassemia, result from a lesion in transcription, or early posttranscriptional processes; the defect appears to be corrected by restoration of proper translational potential to the mutant mRNA, at least in a gene transfer-expression system in tissue-culture cells.

scholarly journals Expression of a cloned Lepore globin gene

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 168-172
Author(s):  
C Dobkin ◽  
J Clyne ◽  
A Metzenberg ◽  
A Bank
Keyword(s):  
Hela Cells ◽  
Molecular Mechanisms ◽  
Globin Gene ◽  
Northern Blotting ◽  
Beta Globin ◽  
Globin Mrna ◽  
S1 Nuclease ◽  
Dna Library ◽  
Phage Dna

Lepore globin is synthesized in markedly diminished amounts (approximately 10% to 15% of normal beta-globin) in human erythroid cells. To study the molecular mechanisms responsible for the diminished biosynthesis of Lepore globin, the Lepore-Boston gene was cloned from a charon phage DNA library and expressed in HeLa cells. Northern blotting and S1 nuclease analyses indicated that the Lepore gene produced less globin mRNA than a beta-gene and more than a delta-gene. The results indicate that expression of the Lepore-Boston gene in HeLa cells is reduced to an extent comparable to that seen in erythroid precursors in vivo. This indicates that the decrease in Lepore globin gene transcription is due to the delta-nucleotide sequences either in the 5′ flanking region or within this gene.

scholarly journals Expression of a cloned Lepore globin gene

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 168-172 ◽  
Author(s):  
C Dobkin ◽  
J Clyne ◽  
A Metzenberg ◽  
A Bank
Keyword(s):  
Hela Cells ◽  
Molecular Mechanisms ◽  
Globin Gene ◽  
Northern Blotting ◽  
Beta Globin ◽  
Globin Mrna ◽  
S1 Nuclease ◽  
Dna Library ◽  
Phage Dna

Abstract Lepore globin is synthesized in markedly diminished amounts (approximately 10% to 15% of normal beta-globin) in human erythroid cells. To study the molecular mechanisms responsible for the diminished biosynthesis of Lepore globin, the Lepore-Boston gene was cloned from a charon phage DNA library and expressed in HeLa cells. Northern blotting and S1 nuclease analyses indicated that the Lepore gene produced less globin mRNA than a beta-gene and more than a delta-gene. The results indicate that expression of the Lepore-Boston gene in HeLa cells is reduced to an extent comparable to that seen in erythroid precursors in vivo. This indicates that the decrease in Lepore globin gene transcription is due to the delta-nucleotide sequences either in the 5′ flanking region or within this gene.

scholarly journals Specific globin mRNAs in human erythroleukemia (K562) cells

Blood ◽  
1984 ◽  
Vol 63 (1) ◽  
pp. 195-200 ◽  
Author(s):  
CW Miller ◽  
K Young ◽  
D Dumenil ◽  
BP Alter ◽  
JM Schofield ◽  
...  
Keyword(s):  
Gene Transcription ◽  
Marked Decrease ◽  
Globin Gene ◽  
K562 Cells ◽  
Dna Probes ◽  
The Other ◽  
Beta Globin ◽  
Globin Mrna ◽  
Mrna Accumulation

Abstract Specific globin mRNA accumulation was quantitated in several lines of K562 cells in the absence and the presence of hemin. Using specific cloned DNA probes, the amounts of zeta, alpha, epsilon and gamma mRNAs were shown to be increased 2–3-fold in the presence of 20 microM hemin. No delta- or beta-globin mRNAs were detectable in any of the lines. In one line, Bos, there was a marked decrease in epsilon-globin mRNA, which increased with hemin, although still to much lower levels than in the other lines. The decreased epsilon-globin mRNA accumulation in Bos is shown to be due to decreased epsilon-globin gene transcription.

scholarly journals A role for DIS3L2 over human nonsense-mediated mRNA decay targets

2019 ◽  
Author(s):  
Paulo J. da Costa ◽  
Juliane Menezes ◽  
Margarida Saramago ◽  
Juan F. García-Moreno ◽  
Hugo A. Santos ◽  
...  
Keyword(s):  
Translation Termination ◽  
Full Length ◽  
Nonsense Mediated Decay ◽  
Endonucleolytic Cleavage ◽  
Nonsense Mediated Mrna Decay ◽  
Translation Termination Codon ◽  
Life Analysis ◽  
Mrna Half Life ◽  
Premature Translation Termination Codon

ABSTRACTThe nonsense-mediated decay (NMD) pathway selectively degrades mRNAs carrying a premature translation-termination codon but also regulates the abundance of a large number of physiological mRNAs that encode full-length proteins. In human cells, NMD-targeted mRNAs are degraded by endonucleolytic cleavage and exonucleolytic degradation from both 5’ and 3’ ends. This is done by a process not yet completely understood that recruits decapping and 5’-to-3’ exonuclease activities, as well as deadenylating and 3’-to-5’ exonuclease exosome activities. In yeast, DIS3/Rrp44 protein is the catalytic subunit of the exosome, but in humans, there are three known paralogues of this enzyme: DIS3, DIS3L1, and DIS3L2. DIS3L1 and DIS3L2 exoribonucleases localize in the same compartment where NMD occurs, but little is known about their role in this process. In order to unveil the role of DIS3L2 in NMD, here we show that some NMD-targets accumulate in DIS3L2-depleted cells. mRNA half-life analysis further supports that these NMD-targets are in fact DIS3L2 substrates. Besides, we observed that DIS3L2 acts over full-length transcripts, through a process that also involves UPF1. Moreover, DIS3L2-mediated decay is dependent on the activity of the terminal uridylyl transferases Zcchc6/11 (TUT7/4). Together, our findings establish a role for DIS3L2 and uridylation in NMD.

scholarly journals Fetal Hemoglobin Inducers from the Natural World: A Novel Approach for Identification of Drugs for the Treatment of β-Thalassemia and Sickle-Cell Anemia

2009 ◽  
Vol 6 (2) ◽  
pp. 141-151 ◽  
Author(s):  
Nicoletta Bianchi ◽  
Cristina Zuccato ◽  
Ilaria Lampronti ◽  
Monica Borgatti ◽  
Roberto Gambari
Keyword(s):  
Plant Extracts ◽  
Fetal Hemoglobin ◽  
Natural World ◽  
Globin Mrna ◽  
Mrna Accumulation ◽  
Lead Compounds ◽  
Novel Approach ◽  
Hereditary Persistence

The objective of this review is to present examples of lead compounds identified from biological material (fungi, plant extracts and agro-industry material) and of possible interest in the field of a pharmacological approach to the therapy of β-thalassemia using molecules able to stimulate production of fetal hemoglobin (HbF) in adults. Concerning the employment of HbF inducers as potential drugs for pharmacological treatment of β-thalassemia, the following conclusions can be reached: (i) this therapeutic approach is reasonable, on the basis of the clinical parameters exhibited by hereditary persistence of fetal hemoglobin patients, (ii) clinical trials (even if still limited) employing HbF inducers were effective in ameliorating the symptoms of β-thalassemia patients, (iii) good correlation ofin vivoandin vitroresults of HbF synthesis and γ-globin mRNA accumulation indicates thatin vitrotesting might be predictive ofin vivoresponses and (iv) combined use of different inducers might be useful to maximize HbF, bothin vitroandin vivo. In this review, we present three examples of HbF inducers from the natural world: (i) angelicin and linear psoralens, contained in plant extracts fromAngelica arcangelicaandAegle marmelos, (ii) resveratrol, a polyphenol found in grapes and several plant extracts and (iii) rapamycin, isolated fromStreptomyces hygroscopicus.

scholarly journals Regulation of beta-globin mRNA accumulation by heme in dimethyl sulfoxide (DMSO)-sensitive and DMSO-resistant murine erythroleukemia cells

Blood ◽  
1994 ◽  
Vol 83 (6) ◽  
pp. 1662-1667 ◽  
Author(s):  
Y Fukuda ◽  
H Fujita ◽  
L Garbaczewski ◽  
S Sassa
Keyword(s):  
Dimethyl Sulfoxide ◽  
Erythroid Differentiation ◽  
Mrna Levels ◽  
Beta Globin ◽  
Globin Mrna ◽  
Mrna Accumulation ◽  
Erythroleukemia Cells ◽  
Dmso Treatment ◽  
Free Heme ◽  
Murine Erythroleukemia

The level of mRNA encoding beta-globin was examined in dimethyl sulfoxide (DMSO)-sensitive (DS), and DMSO-resistant (DR) murine erythroleukemia (MEL) cells. DR cells lack erythroid-specific delta- aminolevulinate (ALA) synthase (AL-AS-E), and fail to undergo erythroid differentiation following treatment with DMSO. Treatment of cells with DMSO markedly increased ALAS-E mRNA in DS cells, while the same treatment downregulated the nonspecific ALA synthase (ALAS-N) mRNA levels in both DS and DR cells. The levels of beta-globin mRNA, heme content, and hemoglobin in DS cells increased, while those in DR cells decreased following treatment with DMSO. Treatment of DR cells with hemin caused an increase in beta-globin mRNA and hemoglobin, and partially restored the DMSO-mediated suppression of beta-globin mRNA and hemoglobin synthesis. DMSO treatment decreased heme oxygenase (HO) mRNA in hemin-treated DS cells, but not in hemin-treated DR cells. These findings indicate that heme is necessary for accumulation of the beta-globin transcript during erythroid differentiation, and that hemin- mediated HO induction becomes markedly downregulated in differentiated erythroid cells, presumably because less free heme is available for HO induction by a greater demand for the synthesis of hemoglobin.

scholarly journals Nonsense codon mutations in the terminal exon of the beta-globin gene are not associated with a reduction in beta-mRNA accumulation: a mechanism for the phenotype of dominant beta-thalassemia

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2031-2037 ◽  
Author(s):  
GW Hall ◽  
S Thein
Keyword(s):  
Globin Gene ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Terminal Exon ◽  
Mrna Accumulation ◽  
Beta Globin Gene ◽  
Globin Chains ◽  
Nonsense Codon ◽  
Nonsense Codons

We present in vivo evidence that there is no reduction in beta-mRNA accumulation in patients with nonsense codons in the terminal exon of the beta-globin gene. Using reverse transcriptase/polymerase chain reaction (RT-PCR), beta-globin cDNA was isolated from the reticulocytes of individuals heterozygous for nonsense codon mutations in exons II and III of the beta-globin gene. Clinically asymptomatic individuals heterozygous for mutations causing premature termination of translation in exon II [beta(0)39(C-T) and F/S71/72(+A)] were found to have almost no mutant beta-cDNA, whereas patients with nonsense codon mutations in exon III [beta 121(G-T) and beta 127(C-T)] with the clinical phenotype of thalassemia intermedia had comparable levels of mutant and normal beta-cDNA. Translation of the mutant beta-mRNA from patients with nonsense codon mutations in exon III would give rise to truncated beta- globin chains, which could explain the more severe phenotype seen in these individuals.

scholarly journals Determinants that contribute to cytoplasmic stability of human c-fos and beta-globin mRNAs are located at several sites in each mRNA.

1988 ◽  
Vol 8 (8) ◽  
pp. 3244-3250 ◽  
Author(s):  
K S Kabnick ◽  
D E Housman
Keyword(s):  
Half Life ◽  
Untranslated Region ◽  
Calf Serum ◽  
Beta Globin ◽  
Mrna Synthesis ◽  
Globin Mrna ◽  
Mrna Species ◽  
Quiescent Cells ◽  
The Stability ◽  
Mrna Half Life

We have analyzed the contributions to cytoplasmic stability in an mRNA species with a very short half-life (human c-fos) and an mRNA species with a very long half-life (human beta-globin). When the human c-fos promoter was used to drive the expression of human c-fos, beta-globin, and chimeric DNAs between c-fos and beta-globin in transfected cells, a pulse of mRNA synthesis was obtained following induction of transcription by refeeding quiescent cells with medium containing 15% calf serum. The mRNA half-life was determined by using Northern (RNA) blot analysis of mRNAs prepared at various times following the pulse of transcription. Under these conditions human c-fos mRNA exhibited a half-life of 6.6 min and human beta-globin mRNA exhibited a half-life of 17.5 h. Replacement of the 3' end of the c-fos mRNA with the 3' end of the beta-globin mRNA increased the half-life of the resultant RNA from 6.6 to 34 min. The reciprocal chimera had a half-life of 34.6 min compared with the 17.5-h half-life of beta-globin mRNA. These results suggest that sequences which make a major contribution to mRNA stability reside in the 3' end of either or both molecules. A chimera in which the 5' untranslated region of globin was replaced by part of the 5' untranslated region of fos led to destabilization of the encoded mRNA. This construct produced an mRNA with a half-life of 6.8 h instead of the 17.5-h half-life of globin. This result suggests that additional determinants of stability reside in the 5' end of these mRNA molecules. Substitution of part of the 5' untranslated region of fos by the 5' untranslated region of beta-globin yielded an mRNA with stability similar to fos mRNA. These results suggest that interactions among sequences within each mRNA contribute to the stability of the respective molecules.

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