scholarly journals The half-life of c-myc mRNA in growing and serum-stimulated cells: influence of the coding and 3' untranslated regions and role of ribosome translocation.

1994 ◽  
Vol 14 (3) ◽  
pp. 2119-2128 ◽  
Author(s):  
D J Herrick ◽  
J Ross
Keyword(s):  
Hela Cells ◽  
Half Life ◽  
Untranslated Region ◽  
Stop Codon ◽  
Beta Globin ◽  
Coding Region ◽  
3T3 Cells ◽  
Globin Mrna ◽  
Gene Encoding ◽  
Sequence Elements

c-myc mRNA contains at least two discrete sequence elements that account for its short half-life, one in the 3' untranslated region and the other in the carboxy-terminal coding region (coding-region determinant). To investigate the function of each determinant, one or both were fused in frame to portions of a gene encoding long-lived beta-globin mRNA. Each chimeric gene was stably transfected into HeLa and NIH 3T3 cells and was transcribed from a constitutive cytomegalovirus promoter or from a serum-regulated c-fos promoter, respectively. The steady-state levels of the chimeric mRNAs in exponentially growing HeLa cells were compared, and their half-lives were measured by two independent methods: (i) in actinomycin D-treated HeLa cells and (ii) after serum addition to starved 3T3 cells. By each method, mRNAs containing either instability determinant were less stable than beta-globin mRNA. mRNA containing only the c-myc 3' untranslated region was not significantly more stable than mRNA with both determinants. In a cell-free mRNA decay system containing polysomes from transfected HeLa cells, mRNA containing the coding-region determinant was destabilized by addition of a specific RNA competitor, whereas mRNA containing only the 3' untranslated region was unaffected. When a stop codon was inserted upstream of the coding-region determinant, the chimeric mRNA was stabilized approximately twofold. These and other data suggest that degradation involving the coding-region determinant occurs most efficiently when ribosomes are translating the determinant.

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.

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.

Polyomavirus enhancer contains multiple redundant sequence elements that activate both DNA replication and gene expression

1985 ◽  
Vol 5 (4) ◽  
pp. 649-658
Author(s):  
G M Veldman ◽  
S Lupton ◽  
R Kamen
Keyword(s):  
Dna Replication ◽  
Globin Gene ◽  
Mrna Levels ◽  
Beta Globin ◽  
Transcriptional Enhancer ◽  
Globin Mrna ◽  
Enhancer Region ◽  
Sequence Elements ◽  
Multiple Copies ◽  
A Site

Sequences that comprise the 244-base-pair polyomavirus enhancer region are also required in cis for viral DNA replication (Tyndall et al., Nucleic Acids Res. 9:6231-6250, 1981). We have studied the relationship between the sequences that activate replication and those that enhance transcription in two ways. One approach, recently described by de Villiers et al. (Nature [London], 312:242-246, 1984), in which the polyomavirus enhancer region was replaced with other viral or cellular transcriptional enhancers suggested that an enhancer function is required for polyomavirus DNA replication. The other approach, described in this paper, was to analyze a series of deletion mutants that functionally dissect the enhancer region and enabled us to localize four sequence elements in this region that are involved in the activation of replication. These elements, which have little sequence homology, are functionally redundant. Element A (nucleotides 5108 through 5130) was synthesized as a 26-mer with XhoI sticky ends, and one or more copies were introduced into a plasmid containing the origin of replication, but lacking the enhancer region. Whereas one copy of the 26-mer activated replication only to 2 to 5% of the wild-type level, two copies inserted in either orientation completely restored replication. We found that multiple copies of the 26-mer were also active as a transcriptional enhancer by measuring the beta-globin mRNA levels expressed from a plasmid that contained either the polyomavirus enhancer or one or more copies of the 26-mer inserted in a site 3' to the beta-globin gene. We observed a correlation between the number of inserted 26-mers and the level of beta-globin RNA expression.

scholarly journals Long-range RNA interaction of two sequence elements required for endonucleolytic cleavage of human insulin-like growth factor II mRNAs.

1995 ◽  
Vol 15 (1) ◽  
pp. 235-245 ◽  
Author(s):  
W Scheper ◽  
D Meinsma ◽  
P E Holthuizen ◽  
J S Sussenbach
Keyword(s):  
Growth Factor ◽  
Long Range ◽  
Cleavage Site ◽  
Untranslated Region ◽  
Cleavage Product ◽  
Coding Region ◽  
Endonucleolytic Cleavage ◽  
Factor Ii ◽  
Sequence Elements ◽  
Rna Interaction

Human insulin-like growth factor II (IGF-II) mRNAs are subject to site-specific endonucleolytic cleavage in the 3' untranslated region, leading to an unstable 5' cleavage product containing the IGF-II coding region and a very stable 3' cleavage product of 1.8 kb. This endonucleolytic cleavage is most probably the first and rate-limiting step in degradation of IGF-II mRNAs. Two sequence elements within the 3' untranslated region are required for cleavage: element I, located approximately 2 kb upstream of the cleavage site, and element II, encompassing the cleavage site itself. We have identified a stable double-stranded RNA stem structure (delta G = -100 kcal/mol [418.4 kJ/mol]) that can be formed between element I and a region downstream of the cleavage site in element II. This structure is conserved among human, rat, and mouse mRNAs. Detailed analysis of the requirements for cleavage shows that the relative position of the elements is not essential for cleavage. Furthermore, the distance between the coding region and the cleavage site does not affect the cleavage reaction. Mutational analysis of the long-range RNA-RNA interaction shows that not only the double-stranded character but also the sequence of the stable RNA stem is important for cleavage.

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.

F8 genetic analysis strategies when standard approaches fail

2014 ◽  
Vol 34 (02) ◽  
pp. 167-173 ◽  
Author(s):  
B. Pezeshkpoor ◽  
A. Pavlova ◽  
J. Oldenburg ◽  
O. El-Maarri
Keyword(s):  
Genetic Analysis ◽  
Half Life ◽  
Untranslated Region ◽  
Mutation Screening ◽  
Screening Methods ◽  
Haemophilia A ◽  
Splice Sites ◽  
Coding Region ◽  
Coding Regions ◽  
Analysis Strategies

SummaryHaemophilia A is a common X-linked recessive disorder caused by mutations in F8 leading to deficiency or dysfunction of coagulant factor VIII (FVIII). Despite tremendous improvements in mutation screening methods, in a small group of patients with FVIII deficiency suffering from haemophilia A, no DNA change can be found. In these patients, analysis reveals no causal mutations even after sequencing the whole coding region of F8 including the flanking splice sites, as well as the promoter and the 3’ untranslated region (UTR). After excluding the mutations mimicking the haemophilia A phenotype in interacting partners of the FVIII protein affecting the half life and transport of the protein, mutations or rearrangements in non-coding regions of F8 have to be considered responsible for the haemophilia A phenotype.In this review, we present the experiences with molecular diagnosis of such cases and approaches to be applied for mutation negative patients.

scholarly journals Targeting of c-myc and β-globin coding sequences to cytoskeletal-bound polysomes by c-myc 3′ untranslated region

1994 ◽  
Vol 298 (1) ◽  
pp. 143-148 ◽  
Author(s):  
J Hesketh ◽  
G Campbell ◽  
M Piechaczyk ◽  
J M Blanchard
Keyword(s):  
In Situ Hybridization ◽  
Untranslated Region ◽  
Mrna Localization ◽  
Eukaryotic Cells ◽  
Beta Globin ◽  
Globin Mrna ◽  
Directional Information ◽  
Coding Sequences ◽  
Membrane Bound

The influence of the 3′ untranslated region on mRNA localization was investigated by measuring the distribution of myc, beta-globin and hybrid myc-globin mRNAs between free, cytoskeletal-bound and membrane-bound polysomes in cells transfected with either control or chimeric gene constructs. c-myc sequences and beta-globin-coding sequences linked to the myc 3′ untranslated region were present at greatest enrichment in cytoskeletal-bound polysomes. beta-Globin mRNA and myc-coding sequences linked to the beta-globin 3′ untranslated region were recovered largely in the free polysomes. In situ hybridization confirmed that replacement of the c-myc 3′ untranslated region by that of globin caused a relocalization of the mRNA. The results suggest that mRNA localization in differentiated eukaryotic cells depends on a mechanism that involves directional information in the 3′ untranslated region of mRNAs.

scholarly journals Identification of a recently evolved goat embryonic beta-globin pseudogene which retains transcriptional activity in vitro.

1984 ◽  
Vol 4 (10) ◽  
pp. 2120-2127 ◽  
Author(s):  
S G Shapiro ◽  
J B Lingrel
Keyword(s):  
Transcriptional Activity ◽  
Stop Codon ◽  
Globin Gene ◽  
Consensus Sequence ◽  
Duplication Event ◽  
Beta Globin ◽  
Gene Sets ◽  
Sequence Elements ◽  
Group 5

A clone containing the entire goat epsilon V beta-globin gene, which lies downstream from the two tandemly duplicated four-gene sets containing the beta C and beta A genes in the linkage group 5'-epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-3', was isolated, and the sequence of the gene was determined. epsilon V is most homologous to the first gene in each of these sets, epsilon I and epsilon III, and appears to be a third duplicated copy of these genes, possibly the first gene in a third four-gene set. Homology of epsilon V to epsilon I is very high (93.2%) in coding regions, and all transcription, processing, and potential translation consensus sequence elements appear to be present, although the Hogness box of epsilon V is altered compared with that of epsilon I by the deletion of an A(AATAAAA----AATAAA). Nevertheless, epsilon V is clearly a pseudogene as a result of two deletions and one insertion (or insertion-deletion) in its coding sequence, the first of which produces an in-frame stop codon at amino acid 54. Unlike the more highly mutated goat beta-like pseudogene duplicates psi beta X and psi beta Z, epsilon V acquired its defects after the duplication event in which it was created. Its recently acquired defects have left the epsilon V promoter sufficiently conserved to retain transcriptional activity in vitro. The acquisition of defects by this gene may be related to the multiple gene duplications which have created at least five epsilon type genes in the goat beta-globin locus.

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.

scholarly journals Comparison between the Repression Potency of siRNA Targeting the Coding Region and the 3′-Untranslated Region of mRNA

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Ching-Fang Lai ◽  
Chih-Ying Chen ◽  
Lo-Chun Au
Keyword(s):  
Gene Silencing ◽  
Thermodynamic Stability ◽  
Untranslated Region ◽  
Stop Codon ◽  
Secondary Structures ◽  
Transcriptional Gene Silencing ◽  
Small Interfering Rnas ◽  
Coding Region ◽  
Post Transcriptional Gene Silencing ◽  
Flanking Sequences

Small interfering RNAs (siRNAs) are applied for post-transcriptional gene silencing by binding target mRNA. A target coding region is usually chosen, although the3′-untranslated region (3′-UTR) can also be a target. This study elucidates whether the coding region or3′-UTR elicits higher repression. pFLuc and pRLuc are two reporter plasmids. A segment ofFLucgene was PCR-amplified and inserted behind the stop codon of theRLucgene of the pRLuc. Similarly, a segment ofRLucgene was inserted behind the stop codon ofFLuc. Two siFLuc and two siRLuc were siRNAs designed to target the central portions of these segments. Therefore, the siRNA encountered the same targets and flanking sequences. Results showed that the two siFLuc elicited higher repression when theFLucsegment resided in the coding region. Conversely, the two siRLuc showed higher repression when theRLucsegment was in the3′-UTR. These results indicate that both the coding region and the3′-UTR can be more effective targets. The thermodynamic stability of the secondary structures was analyzed. The siRNA elicited higher repression in the coding region when the target configuration was stable, and needed to be solved by translation. A siRNA may otherwise favor the target at3′-UTR.

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