scholarly journals Nonsense codons in human beta-globin mRNA result in the production of mRNA degradation products.

1992 ◽  
Vol 12 (3) ◽  
pp. 1149-1161 ◽  
Author(s):  
S K Lim ◽  
C D Sigmund ◽  
K W Gross ◽  
L E Maquat
Keyword(s):  
Premature Termination ◽  
Degradation Products ◽  
Mrna Translation ◽  
Mrna Degradation ◽  
Full Length ◽  
Cytoplasmic Process ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Nonsense Codons

Human beta zero-thalassemic beta-globin genes harboring either a frameshift or a nonsense mutation that results in the premature termination of beta-globin mRNA translation have been previously introduced into the germ line of mice (S.-K. Lim, J.J. Mullins, C.-M. Chen, K. Gross, and L.E. Maquat, EMBO J. 8:2613-2619, 1989). Each transgene produces properly processed albeit abnormally unstable mRNA as well as several smaller RNAs in erythroid cells. These smaller RNAs are detected only in the cytoplasm and, relative to mRNA, are longer-lived and are missing sequences from either exon I or exons I and II. In this communication, we show by using genetics and S1 nuclease transcript mapping that the premature termination of beta-globin mRNA translation is mechanistically required for the abnormal RNA metabolism. We also provide evidence that generation of the smaller RNAs is a cytoplasmic process: the 5' ends of intron 1-containing pre-mRNAs were normal, the rates of removal of introns 1 and 2 were normal, and studies inhibiting RNA synthesis with actinomycin D demonstrated a precursor-product relationship between full-length mRNA and the smaller RNAs. In vivo, about 50% of the full-length species that undergo decay are degraded to the smaller RNAs and the rest are degraded to undetectable products. Exposure of erythroid cells that expressed a normal human beta-globin transgene to either cycloheximide or puromycin did not result in the generation of the smaller RNAs. Therefore, a drug-induced reduction in cellular protein synthesis does not reproduce this aspect of cytoplasmic mRNA metabolism. These data suggest that the premature termination of beta-globin mRNA translation in either exon I or exon II results in the cytoplasmic generation of discrete mRNA degradation products that are missing sequences from exon I or exons I and II. Since these degradation products appear to be the same for all nonsense codons tested, there is no correlation between the position of translation termination and the sites of nucleolytic cleavage.

Nonsense codons in human beta-globin mRNA result in the production of mRNA degradation products

1992 ◽  
Vol 12 (3) ◽  
pp. 1149-1161
Author(s):  
S K Lim ◽  
C D Sigmund ◽  
K W Gross ◽  
L E Maquat
Keyword(s):  
Premature Termination ◽  
Degradation Products ◽  
Mrna Translation ◽  
Mrna Degradation ◽  
Full Length ◽  
Cytoplasmic Process ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Nonsense Codons

Human beta zero-thalassemic beta-globin genes harboring either a frameshift or a nonsense mutation that results in the premature termination of beta-globin mRNA translation have been previously introduced into the germ line of mice (S.-K. Lim, J.J. Mullins, C.-M. Chen, K. Gross, and L.E. Maquat, EMBO J. 8:2613-2619, 1989). Each transgene produces properly processed albeit abnormally unstable mRNA as well as several smaller RNAs in erythroid cells. These smaller RNAs are detected only in the cytoplasm and, relative to mRNA, are longer-lived and are missing sequences from either exon I or exons I and II. In this communication, we show by using genetics and S1 nuclease transcript mapping that the premature termination of beta-globin mRNA translation is mechanistically required for the abnormal RNA metabolism. We also provide evidence that generation of the smaller RNAs is a cytoplasmic process: the 5' ends of intron 1-containing pre-mRNAs were normal, the rates of removal of introns 1 and 2 were normal, and studies inhibiting RNA synthesis with actinomycin D demonstrated a precursor-product relationship between full-length mRNA and the smaller RNAs. In vivo, about 50% of the full-length species that undergo decay are degraded to the smaller RNAs and the rest are degraded to undetectable products. Exposure of erythroid cells that expressed a normal human beta-globin transgene to either cycloheximide or puromycin did not result in the generation of the smaller RNAs. Therefore, a drug-induced reduction in cellular protein synthesis does not reproduce this aspect of cytoplasmic mRNA metabolism. These data suggest that the premature termination of beta-globin mRNA translation in either exon I or exon II results in the cytoplasmic generation of discrete mRNA degradation products that are missing sequences from exon I or exons I and II. Since these degradation products appear to be the same for all nonsense codons tested, there is no correlation between the position of translation termination and the sites of nucleolytic cleavage.

scholarly journals Equal stabilities of normal beta globin and nontranslatable beta0 -39 thalassemic transcripts in cell-free extracts

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 293-300 ◽  
Author(s):  
CA Stolle ◽  
MS Payne ◽  
EJ Jr Benz
Keyword(s):  
Beta Globin ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Dna Templates ◽  
Relative Stabilities ◽  
The Stability

Abstract Patients with beta zero thalassemia arising from premature terminator codon mutations in the gene for beta globin do not produce beta globin protein; these individuals also exhibit a decreased amount of beta globin mRNA in their erythroid cells. The absence of beta globin protein is readily explained by the inability of the beta zero-39 mRNA to be translated. The decrease in beta globin mRNA has been attributed to either decreased cytoplasmic stability of the nontranslatable decreased cytoplasmic stability of the nontranslatable mRNA or to an undefined nuclear lesion. To compare directly the relative stabilities of normal and beta zero-39 thalassemic globin transcripts, we prepared normal and thalassemic beta globin pre-mRNAs and mRNAs using cloned DNA templates and the SP6 promoter-polymerase system. The stability of the transcripts was assessed by incubation in various cell-free extracts. Our results indicate that although the stabilities of the beta globin transcripts varied considerably from one extract to another the stabilities of the beta zero-39 thalassemic pre-mRNAs and mRNAs were equal to those of normal beta globin mRNAs in every extract tested.

scholarly journals Functional characterization of a non-AUUUA AU-rich element from the c-jun proto-oncogene mRNA: evidence for a novel class of AU-rich elements.

1996 ◽  
Vol 16 (4) ◽  
pp. 1490-1499 ◽  
Author(s):  
S S Peng ◽  
C Y Chen ◽  
A B Shyu
Keyword(s):  
Functional Characterization ◽  
Structural Features ◽  
Mrna Degradation ◽  
Beta Globin ◽  
Globin Mrna ◽  
Transcription Inhibitors ◽  
Tightly Coupled ◽  
Underlying Mechanisms ◽  
Necessary And Sufficient ◽  
Domain I

AU-rich RNA-destabilizing elements (AREs) found in the 3' untranslated regions of many labile mRNAs encoding proto-oncoproteins and cytokines generally contain (i) one or more copies of the AUUUA pentanucleotide and (ii) a high content of uridylate and sometimes also adenylate residues. Recently, we have identified a potent ARE from the 3' untranslated region of c-jun proto-oncogene mRNA that does not contain the AUUUA motif. In an attempt to further our understanding of the general principles underlying mechanisms by which AREs direct rapid and selective mRNA degradation, in this study we have characterized the functionally important structural features and properties of this non-AUUUA ARE. Like AUUUA-containing AREs, this non-AUUUA ARE directs rapid shortening of the poly(A) tail as a necessary first step for mRNA degradation. It can be further dissected into three structurally and functionally distinct regions, designated domains I, II, and III. None of three domains alone is able to significantly destabilize the stable beta-globin mRNA. The two unlinked domains, I and III, together are necessary and sufficient for specifying the full destabilizing function of this non-AUUUA ARE. Domain II appears functionally dispensable but can partially substitute for domain I. Domain swaps made between the c-jun non-AUUUA and the c-fos AUUUA-containing AREs reveal that the two AREs, while sharing no sequence homology, appear to contain sequence domains that are structurally distinct but functionally overlapping and exchangeable. These data support the idea that the ultimate destabilizing function of an individual ARE is determined by its own unique combination of structurally distinct and functionally interdependent domains. Our polysome profile studies show tha the destabilizing function of the c-jun non-AUUUA ARE does not require any active transit by ribosomes of the mRNA bearing it, further corroborating that the destabilizing function of AREs is not tightly coupled to ongoing translation by ribosomes. Moreover, unlike AUUUA-containing AREs, the c-jun ARE is insensitive to blockage of its effects by addition of transcription inhibitors. Thus, our data provide further evidence for the existence of a novel class of ARE with unique properties.

scholarly journals The stability of human beta-globin mRNA is dependent on structural determinants positioned within its 3' untranslated region

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 5314-5323 ◽  
Author(s):  
JE Russell ◽  
SA Liebhaber
Keyword(s):  
Mrna Stability ◽  
Globin Gene ◽  
Structural Features ◽  
Structural Basis ◽  
Dependent Manner ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Coding Region ◽  
Globin Mrna ◽  
Alpha Globin

Controls that act at both transcriptional and posttranscriptional levels assure that globin genes are highly expressed in developing erythroid cells. The extraordinary stabilities of alpha- and beta- globin mRNAs permit globin proteins to accumulate to substantial levels in these cells, even in the face of physiologic transcriptional silencing. Structural features that determine alpha-globin mRNA stability have recently been identified within its 3′UTR; in contrast, the structural features that determine beta-globin mRNA stability remain obscure. The current study begins to define the structural basis for beta-globin mRNA stability. Two tandem antitermination mutations are introduced into the wild-type human beta-globin gene that permit ribosomes to read into the 3′UTR of the encoded beta-globin mRNA. The readthrough beta-globin mRNA is destabilized in cultured erythroid cells, indicating that, as in human alpha-globin mRNA, an unperturbed 3′UTR is crucial to maintaining mRNA stability. Additional experiments show that the beta-globin and alpha-globin mRNA 3′UTRs provide equivalent levels of stability to a linked beta-globin mRNA coding region, suggesting a parallel in their functions. However, destabilization of the antiterminated beta-globin mRNA is independent of active translation into the 3′UTR, whereas translation into the alpha-globin mRNA 3′UTR destabilizes a linked beta-globin coding region in a translationally dependent manner. This indicates that the alpha- and beta-globin 3′UTRs may stabilize linked mRNAs through distinct mechanisms. Finally, it is shown that neither of the two mutations that, in combination, destabilize the beta-globin mRNA have any effect on beta-globin mRNA stability when present singly, suggesting potential redundancy of stabilizing elements. In sum, the current study shows that a functionally intact beta-globin mRNA 3′UTR is crucial to maintaining beta-globin mRNA stability and provides a level of stability that is functionally equivalent to, although potentially mechanistically distinct from, the previously characterized alpha- globin mRNA 3′UTR stability element.

High-efficiency cloning of full-length cDNA

1982 ◽  
Vol 2 (2) ◽  
pp. 161-170
Author(s):  
H Okayama ◽  
P Berg
Keyword(s):  
Plasmid Dna ◽  
High Efficiency ◽  
Full Length ◽  
Mrna Sequence ◽  
Beta Globin ◽  
Cdna Synthesis ◽  
Globin Mrna ◽  
Full Length Cdna ◽  
Coding Regions ◽  
Entire Nucleotide Sequence

A widely recognized difficulty of presently used methods for cDNA cloning is obtaining cDNA segments that contain the entire nucleotide sequence of the corresponding mRNA. The cloning procedure described here mitigates this shortcoming. Of the 10(5) plasmid-cDNA recombinants obtained per microgram of rabbit reticulocyte mRNA, about 10% contained a complete alpha- of beta-globin mRNA sequence, and at least 30 to 50%, but very likely more, contained the entire globin coding regions. We attribute the high efficiency of cloning full- or nearly full-length cDNA to (i) the fact that the plasmid DNA vector itself serves as the primer for first- and second-strand cDNA synthesis, (ii) the lack of any nuclease treatment of the products, and (iii) the fact that one of the steps in the procedure results in preferential cloning of recombinants with full-length cDNA's over those with truncated cDNA's.

scholarly journals Two novel beta-thalassemia mutations in the 5' and 3' noncoding regions of the beta-globin gene [see comments]

Blood ◽  
1992 ◽  
Vol 79 (5) ◽  
pp. 1342-1346 ◽  
Author(s):  
SP Cai ◽  
B Eng ◽  
WH Francombe ◽  
NF Olivieri ◽  
AG Kendall ◽  
...  
Keyword(s):  
Premature Termination ◽  
Globin Gene ◽  
Polyadenylation Signal ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Globin Mrna ◽  
Noncoding Regions ◽  
Beta Globin Gene ◽  
Italian Family ◽  
Irish Family

Abstract Two novel beta-thalassemia mutations are described. The first mutation, found in an Italian family, is a G----A substitution in nucleotide (nt) +22 relative to the beta-globin gene Cap site. This mutation creates a cryptic ATG initiation codon, the utilization of which for translation would result in premature termination 36 bp 3′ downstream. The second mutation, found in an Irish family, is a T----C substitution in nt +1570, or 12 bp 5′ upstream of the AATAAA polyadenylation signal in the 3′ noncoding region. It is postulated that this mutation leads to destabilization of the encoded beta-globin mRNA.

scholarly journals Equal stabilities of normal beta globin and nontranslatable beta0 -39 thalassemic transcripts in cell-free extracts

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 293-300
Author(s):  
CA Stolle ◽  
MS Payne ◽  
EJ Jr Benz
Keyword(s):  
Beta Globin ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Dna Templates ◽  
Relative Stabilities ◽  
The Stability

Patients with beta zero thalassemia arising from premature terminator codon mutations in the gene for beta globin do not produce beta globin protein; these individuals also exhibit a decreased amount of beta globin mRNA in their erythroid cells. The absence of beta globin protein is readily explained by the inability of the beta zero-39 mRNA to be translated. The decrease in beta globin mRNA has been attributed to either decreased cytoplasmic stability of the nontranslatable decreased cytoplasmic stability of the nontranslatable mRNA or to an undefined nuclear lesion. To compare directly the relative stabilities of normal and beta zero-39 thalassemic globin transcripts, we prepared normal and thalassemic beta globin pre-mRNAs and mRNAs using cloned DNA templates and the SP6 promoter-polymerase system. The stability of the transcripts was assessed by incubation in various cell-free extracts. Our results indicate that although the stabilities of the beta globin transcripts varied considerably from one extract to another the stabilities of the beta zero-39 thalassemic pre-mRNAs and mRNAs were equal to those of normal beta globin mRNAs in every extract tested.

scholarly journals Two novel beta-thalassemia mutations in the 5' and 3' noncoding regions of the beta-globin gene [see comments]

Blood ◽  
1992 ◽  
Vol 79 (5) ◽  
pp. 1342-1346 ◽  
Author(s):  
SP Cai ◽  
B Eng ◽  
WH Francombe ◽  
NF Olivieri ◽  
AG Kendall ◽  
...  
Keyword(s):  
Premature Termination ◽  
Globin Gene ◽  
Polyadenylation Signal ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Globin Mrna ◽  
Noncoding Regions ◽  
Beta Globin Gene ◽  
Italian Family ◽  
Irish Family

Two novel beta-thalassemia mutations are described. The first mutation, found in an Italian family, is a G----A substitution in nucleotide (nt) +22 relative to the beta-globin gene Cap site. This mutation creates a cryptic ATG initiation codon, the utilization of which for translation would result in premature termination 36 bp 3′ downstream. The second mutation, found in an Irish family, is a T----C substitution in nt +1570, or 12 bp 5′ upstream of the AATAAA polyadenylation signal in the 3′ noncoding region. It is postulated that this mutation leads to destabilization of the encoded beta-globin mRNA.

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