scholarly journals Co-operation of the 5′ and 3′ untranslated regions of ornithine decarboxylase mRNA and inhibitory role of its 3′ untranslated region in regulating the translational efficiency of hybrid RNA species via cellular factor(s)

1997 ◽  
Vol 326 (2) ◽  
pp. 361-367 ◽  
Author(s):  
Erna C. LORENZINI ◽  
Immo E. SCHEFFLER
Keyword(s):  
Ornithine Decarboxylase ◽  
Untranslated Region ◽  
Expression Vectors ◽  
Serum Starvation ◽  
Translational Efficiency ◽  
Cellular Factor ◽  
Mrna Species ◽  
Inhibitory Role ◽  
Partial Release

The 5′ untranslated region (UTR) has an inhibitory role in the translatability of ornithine decarboxylase (ODC) mRNA and of hybrid mRNA species, whereas the ODC 3′ UTR causes a partial release of this inhibition. We designed experiments to explore whether the co-operation between ODC 5′ UTR and 3′ UTR in the translational regulation is due to a direct interaction of those sequences or whether it is mediated by their interaction with cellular factor(s). We stably transfected Chinese hamster ovary (CHO)-K1 cells and transiently transfected COS-1 cells with expression vectors carrying different chimaeric DNAs having the luciferase (LUC) coding sequence as reporter gene, the ODC 5′ UTR or the ODC 3′ UTR, or both, in the appropriate positions. We compared the results obtained by assaying the LUC activities of both transfected cell lines with each chimaeric DNA with those observed by translating the hybrid RNAs in a translation system in vitro. When the ODC 3′ UTR was present, we observed a partial release of the translation inhibition owing to the ODC 5′ UTR only in vivo. The releasing effect was restored in vitro by the addition of cytoplasmic extracts from wild-type CHO-K1 or COS-1 cells, prepared 2 and 8 h after their release from serum starvation. We also observed a partial inhibition of the translatability of the hybrid RNA owing to the presence of the ODC 3′ UTR itself; the translational efficiency could be rescued by cell extract from 8 h serum-stimulated cells. The co-operation between the ODC-UTRs might be mediated by factors expressed by cells during particular phases of the cell cycle. Excess copies of the ODC-UTRs, expressed in trans, could compete in binding limited amounts of such regulatory factors and remove them from interaction with the endogenous ODC mRNA. This phenomenon should be reflected by modifications of the kinetics of ODC and/or LUC activities during serum stimulation. The overexpression of the ODC 3′ UTR determined an increase in both endogenous ODC activity and LUC activity. Moreover, in the transfectants expressing the hybrid RNA species bearing the ODC 3′ UTR the basal ODC activity is higher than that observed in control cells. We suggest that excess copies of the ODC 3′ UTR mis-regulate the endogenous ODC translatability, probably by tying up regulatory molecules expressed by cells in limited amounts and sequestering them from the ODC mRNA species they should interact with.

scholarly journals Reduced translation efficiency due to novel splicing variants in 5′ untranslated region and identification of novel cis-regulatory elements in canine and human BRCA2

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yasunaga Yoshikawa ◽  
Hajime Kozuma ◽  
Masami Morimatsu ◽  
Kaori Sugawara ◽  
Koichi Orino
Keyword(s):  
Homologous Recombination ◽  
Untranslated Region ◽  
Cultured Cells ◽  
Regulatory Elements ◽  
Expression Level ◽  
Serum Starvation ◽  
Translational Efficiency ◽  
Translation Efficiency ◽  
Intron 1 ◽  
Splicing Variants

Abstract Background Breast cancer 2, early onset (BRCA2) is a tumor suppressor gene. The protein encoded by this gene plays an important role in homologous recombination (HR)-mediated DNA repair. Deleterious mutations in BRCA2 and downregulation of its expression have been associated with tumorigenesis in dogs and humans. Thus, regulation of BRCA2 expression level is important for maintaining homeostasis in homologous recombination. Results In this study, the mechanisms that regulate the expression of BRCA2 were proposed. Novel splicing variants were identified in the 5′ untranslated region (UTR) of canine and human BRCA2 in canine testis, canine ovary, and canine and human cultured cell lines. In cultured cells, the ratio of BRCA2 splicing variants at the 5′ UTR was altered by serum starvation. These novel splicing variants, excluding one of the canine splicing variants, were found to reduce the translational efficiency. Additionally, the DNA sequence in human BRCA2 intron 1 harbored novel cis-regulatory elements. Three silencer and two enhancer cis-regulatory elements were identified in human BRCA2 intron 1. Conclusions This study demonstrates that BRCA2 expression level is regulated via 5′ UTR splicing variants and that the BRCA2 intron 1 region harbors cis-regulatory elements.

Control of Na+-K+-ATPase β1-subunit expression: role of 3′-untranslated region

2004 ◽  
Vol 286 (3) ◽  
pp. C580-C585 ◽  
Author(s):  
Yvonne Shao ◽  
Faramarz Ismail-Beigi
Keyword(s):  
Untranslated Region ◽  
Ventricular Myocardium ◽  
Distal Segment ◽  
In Vitro Translation ◽  
Luciferase Expression ◽  
Translational Efficiency ◽  
Stem Loop ◽  
Subunit Expression

Using in vitro translation and cell transfection assays, we previously demonstrated that the Na+-K+-ATPase β1 mRNA species containing its longest 3′-untranslated region (UTR) exhibited the lowest translational efficiency. Here, employing deletions and in vivo expression assays, using direct injection of plasmids into rat ventricular myocardium, we identified a 143-nt segment located in the distal 3′-UTR of β1 mRNA that was associated with decreased luciferase expression; interestingly, this segment contains three AUUUA motifs. Using RNA-protein binding assays and UV cross-linking of cRNA with cytosolic proteins of rat heart, we identified an ∼38-kDa protein that specifically bound to the cRNA encoding the 143-nt segment of β1 mRNA 3′-UTR. Mutation of three nucleotides located in the middle region of the 143-nt segment, which was predicted to greatly disrupt a putative stem-loop structure of the cRNA in this region, was associated with reduced binding of the mutated cRNA to the protein migrating at ∼38 kDa. The cRNA encoding a segment of cyclooxygenase-2 mRNA 3′-UTR containing six AUUUA sequences did not bind the protein migrating at ∼38 kDa and did not compete with the binding of the wild-type 143-nt β1 cRNA to the protein. The above results suggest that the 143-nt segment in the distal segment of the 3′-UTR of β1 mRNA may play an important role in the control of β1-subunit expression.

scholarly journals Reduced translation efficiency due to novel splicing variants in 5′ untranslated region and identification of novel cis-regulatory elements in canine and human BRCA2

2020 ◽  
Author(s):  
Yasunaga Yoshikawa ◽  
Hajime Kozuma ◽  
Masami Morimatsu ◽  
Kaori Sugawara ◽  
Koichi Orino
Keyword(s):  
Untranslated Region ◽  
Cultured Cells ◽  
Regulatory Elements ◽  
Suppressor Gene ◽  
Expression Level ◽  
Serum Starvation ◽  
Translational Efficiency ◽  
Translation Efficiency ◽  
Intron 1 ◽  
Splicing Variants

Abstract Background: Breast cancer 2, early onset (BRCA2) is a tumor suppressor gene. The protein encoded by this gene plays an important role in homologous recombination (HR)-mediated DNA repair. Deleterious mutations in BRCA2 and downregulation of its expression have been associated with tumorigenesis in dogs and humans. Thus, regulation of BRCA2 expression level is important for maintaining homeostasis in homologous recombination.Results: In this study, the mechanisms that regulate the expression of BRCA2 were proposed. Novel splicing variants were identified in the 5′ untranslated region (UTR) of canine and human BRCA2 in canine testis, canine ovary, and canine and human cultured cell lines. In cultured cells, the ratio of BRCA2 splicing variants at the 5′ UTR was altered by serum starvation. These novel splicing variants, excluding one of the canine splicing variants, were found to reduce the translational efficiency. Additionally, the DNA sequence in human BRCA2 intron 1 harbored novel cis-regulatory elements. Three silencer and two enhancer cis-regulatory elements were identified in human BRCA2 intron 1.Conclusions: This study demonstrates that BRCA2 expression level is regulated via 5′ UTR splicing variants and that the BRCA2 intron 1 region harbors cis-regulatory elements.

scholarly journals Control of the translational efficiency of beta-F1-ATPase mRNA depends on the regulation of a protein that binds the 3' untranslated region of the mRNA.

1997 ◽  
Vol 17 (9) ◽  
pp. 5255-5268 ◽  
Author(s):  
J M Izquierdo ◽  
J M Cuezva
Keyword(s):  
Untranslated Region ◽  
Fetal Liver ◽  
Mrna Translation ◽  
Full Length ◽  
In Vitro Translation ◽  
Translational Efficiency ◽  
Reporter Construct ◽  
F1 Atpase ◽  
Atpase Gene

The expression of the nucleus-encoded beta-F1-ATPase gene of oxidative phosphorylation is developmentally regulated in the liver at both the transcriptional and posttranscriptional levels. In this study we have analyzed the potential mechanisms that control the cytoplasmic expression of beta-F1-ATPase mRNA during liver development. Remarkably, a full-length 3' untranslated region (UTR) of the transcript is required for its efficient in vitro translation. When the 3' UTR of beta-F1-ATPase mRNA is placed downstream of a reporter construct, it functions as a translational enhancer. In vitro translation experiments with full-length beta-F1-ATPase mRNA and with a chimeric reporter construct containing the 3' UTR of beta-F1-ATPase mRNA suggested the existence of an inhibitor of beta-F1-ATPase mRNA translation in the fetal liver. Electrophoretic mobility shift assays and UV cross-linking experiments allowed the identification of an acutely regulated protein (3'betaFBP) of the liver that binds at the 3' UTR of beta-F1-ATPase mRNA. The developmental profile of 3'betaFBP parallels the reported changes in the translational efficiency of beta-F1-ATPase mRNA during development. Fractionation of fetal liver extracts revealed that the inhibitory activity of beta-F1-ATPase mRNA translation cofractionates with 3'-UTR band-shifting activity. Compared to other tissues of the adult rat, kidney and spleen extracts showed very high expression levels of 3'betaFBP. Translation of beta-F1-ATPase mRNA in the presence of kidney and spleen extracts further supported a translational inhibitory role for 3'betaFBP. Mapping experiments and a deletion mutant of the 3' UTR revealed that the cis-acting element for binding 3'betaFBP is located within a highly conserved region of the 3' UTR of mammalian beta-F1-ATPase mRNAs. Overall, we have identified a mechanism of translational control that regulates the rapid postnatal differentiation of liver mitochondria.

scholarly journals Both the 5' untranslated region and the sequences surrounding the start site contribute to efficient initiation of translation in vitro.

1991 ◽  
Vol 11 (5) ◽  
pp. 2656-2664 ◽  
Author(s):  
D Falcone ◽  
D W Andrews
Keyword(s):  
Untranslated Region ◽  
Leader Sequence ◽  
Limiting Factor ◽  
Translational Efficiency ◽  
Beta Globin ◽  
Rna Sequences ◽  
Coding Regions ◽  
Initiation Of Translation ◽  
Initiation Sequence

The role of RNA sequences in the 5' leader region between the cap site and initiating AUG in mediating translation was examined in vitro. Hybrid mRNAs were synthesized in which the cognate leader sequence was replaced with either optimized or compromised leader sequences, and translational efficiency was measured for six different coding regions. Translation was most efficient with a leader containing the 5' untranslated region from Xenopus beta-globin and an optimized initiation sequence. Compared with the cognate leaders, this hybrid was observed to increase translation of the various coding regions as much as 300-fold. The translational efficiencies of the different coding regions also varied substantially. In contrast to earlier suggestions that increased leader efficiency results from higher affinity of the leader for a limiting factor, our experiments suggest that increased translation from the beta-globin hybrid leader sequence results from more rapid initiation of translation.

scholarly journals Reduced translation efficiency due to novel splicing variants in 5′ untranslated region and identification of novel cis-regulatory elements in canine and human BRCA2

2020 ◽  
Author(s):  
Yasunaga Yoshikawa ◽  
Masami Morimatsu ◽  
Hajime Kozuma ◽  
Kaori Sugawara ◽  
Koichi Orino
Keyword(s):  
Homologous Recombination ◽  
Untranslated Region ◽  
Cultured Cells ◽  
Regulatory Elements ◽  
Expression Level ◽  
Serum Starvation ◽  
Translational Efficiency ◽  
Translation Efficiency ◽  
Intron 1 ◽  
Splicing Variants

Abstract Background Breast cancer 2, early onset (BRCA2) is a tumor suppressor gene. The protein encoded by this gene plays an important role in homologous recombination (HR)-mediated DNA repair. Deleterious mutations in BRCA2 and downregulation of its expression have been associated with tumorigenesis in dogs and humans. Thus, regulation of BRCA2 expression level is important for maintaining homeostasis in homologous recombination. Results In this study, the mechanisms that regulate the expression of BRCA2 were proposed. Novel splicing variants were identified in the 5′ untranslated region (UTR) of canine and human BRCA2 in canine testis, canine ovary, and canine and human cultured cell lines. In cultured cells, the ratio of BRCA2 splicing variants at 5′ UTR was altered by serum starvation. These novel splicing variants, excluding one of the canine splicing variants, were found to reduce the translational efficiency. Additionally, the DNA sequence in human BRCA2 intron 1 harbored novel cis-regulatory elements. Three silencer and two enhancer cis-regulatory elements were identified in human BRCA2 intron 1. Conclusions This study demonstrates that BRCA2 expression level is regulated via 5′ UTR splicing variants and that the BRCA2 intron 1 region harbors cis-regulatory elements.

scholarly journals Reduced translation efficiency due to novel splicing variants in 5′ untranslated region and identification of novel cis-regulatory elements in canine and human BRCA2

2020 ◽  
Author(s):  
Yasunaga Yoshikawa ◽  
Hajime Kozuma ◽  
Masami Morimatsu ◽  
Kaori Sugawara ◽  
Koichi Orino
Keyword(s):  
Homologous Recombination ◽  
Untranslated Region ◽  
Cultured Cells ◽  
Regulatory Elements ◽  
Expression Level ◽  
Serum Starvation ◽  
Translational Efficiency ◽  
Translation Efficiency ◽  
Intron 1 ◽  
Splicing Variants

Abstract Background: Breast cancer 2, early onset (BRCA2) is a tumor suppressor gene. The protein encoded by this gene plays an important role in homologous recombination (HR)-mediated DNA repair. Deleterious mutations in BRCA2 and downregulation of its expression have been associated with tumorigenesis in dogs and humans. Thus, regulation of BRCA2 expression level is important for maintaining homeostasis in homologous recombination.Results: In this study, the mechanisms that regulate the expression of BRCA2 were proposed. Novel splicing variants were identified in the 5′ untranslated region (UTR) of canine and human BRCA2 in canine testis, canine ovary, and canine and human cultured cell lines. In cultured cells, the ratio of BRCA2 splicing variants at the 5′ UTR was altered by serum starvation. These novel splicing variants, excluding one of the canine splicing variants, were found to reduce the translational efficiency. Additionally, the DNA sequence in human BRCA2 intron 1 harbored novel cis-regulatory elements. Three silencer and two enhancer cis-regulatory elements were identified in human BRCA2 intron 1.Conclusions: This study demonstrates that BRCA2 expression level is regulated via 5′ UTR splicing variants and that the BRCA2 intron 1 region harbors cis-regulatory elements.

Both the 5' untranslated region and the sequences surrounding the start site contribute to efficient initiation of translation in vitro

1991 ◽  
Vol 11 (5) ◽  
pp. 2656-2664
Author(s):  
D Falcone ◽  
D W Andrews
Keyword(s):  
Untranslated Region ◽  
Leader Sequence ◽  
Limiting Factor ◽  
Translational Efficiency ◽  
Beta Globin ◽  
Rna Sequences ◽  
Coding Regions ◽  
Initiation Of Translation ◽  
Initiation Sequence

The role of RNA sequences in the 5' leader region between the cap site and initiating AUG in mediating translation was examined in vitro. Hybrid mRNAs were synthesized in which the cognate leader sequence was replaced with either optimized or compromised leader sequences, and translational efficiency was measured for six different coding regions. Translation was most efficient with a leader containing the 5' untranslated region from Xenopus beta-globin and an optimized initiation sequence. Compared with the cognate leaders, this hybrid was observed to increase translation of the various coding regions as much as 300-fold. The translational efficiencies of the different coding regions also varied substantially. In contrast to earlier suggestions that increased leader efficiency results from higher affinity of the leader for a limiting factor, our experiments suggest that increased translation from the beta-globin hybrid leader sequence results from more rapid initiation of translation.

3'-untranslated region of SP-B mRNA mediates inhibitory effects of TPA and TNF-alpha on SP-B expression

1994 ◽  
Vol 267 (1) ◽  
pp. L16-L24 ◽  
Author(s):  
G. S. Pryhuber ◽  
S. L. Church ◽  
T. Kroft ◽  
A. Panchal ◽  
J. A. Whitsett
Keyword(s):  
Growth Hormone ◽  
Untranslated Region ◽  
Human Growth ◽  
Cellular Growth ◽  
Expression Vectors ◽  
Tnf Alpha ◽  
Inhibitory Effects ◽  
Necrosis Factor Alpha

Surfactant protein-B (SP-B) is a small hydrophobic polypeptide that enhances spreading and stability of surfactant phospholipids in the alveolus of the lung. Decreased expression of SP-B is associated with respiratory failure in premature infants and in adult patients with acute respiratory distress syndrome (ARDS). Tumor necrosis factor-alpha (TNF-alpha) and 12-O-tetradecanoylphorbol-13 acetate (TPA) cause ARDS-like lung injury in vivo. Inhibitory effects of TPA and TNF-alpha on SP-B mRNA expression in vitro were mediated by decreased SP-B mRNA stability rather than by decreased rate of SP-B gene transcription. In the present study, a human pulmonary adenocarcinoma cell line, NCI H441-4, was stably transfected with expression vectors consisting of the thymidine kinase (TK) promotor and human growth hormone (hGH) gene, in which the hGH 3'-untranslated region (3'-UTR) was replaced by the 2.0-kb human SP-B cDNA [pTKGH(SP-B2.0)] or the 837-bp human SP-B 3'-UTR [pTKGH(SP-B.837)]. The mRNAs and cellular growth hormone protein generated from the chimeric TKGH(SP-B2.0) and TKGH(SP-B.837) genes were each inhibited by approximately 50% by TPA and TNF-alpha. Dexamethasone decreased the inhibitory effects of TPA and TNF-alpha. The inhibition of steady-state hGH-SP-B mRNA by TPA and TNF-alpha was mediated by a cis-active element located in the 3-UTR region of SP-B mRNA.

scholarly journals The translation in vitro of rat ornithine decarboxylase mRNA is blocked by its 5′ untranslated region in a polyamine-independent way

1991 ◽  
Vol 274 (2) ◽  
pp. 521-526 ◽  
Author(s):  
H Van Steeg ◽  
C T M Van Oostrom ◽  
H M Hodemaekers ◽  
L Peters ◽  
A A M Thomas
Keyword(s):  
Ornithine Decarboxylase ◽  
Translational Control ◽  
Untranslated Region ◽  
Gc Content ◽  
Inhibitory Effect ◽  
Translation Efficiency ◽  
Globin Mrna ◽  
T7 Promoter ◽  
Phage T7

The enzyme ornithine decarboxylase (ODC, EC 4.1.1.17) is believed to play an essential role in the growth and differentiation of cells by regulating the biosynthesis of polyamines. The 5′ untranslated region (5′ UTR) of the ODC mRNA of different species is rather unusual in length and GC content, and may therefore be involved in translational control of ODC protein synthesis. We cloned the rat ODC cDNA downstream of the phage T7 promoter in order to perform transcription/translation studies in vitro. Our results show that the intact 5′ UTR of rat ODC mRNA, which is 303 nt in length, is a potent inhibitor of translation. Efficient synthesis in vitro of ODC protein is obtained when either 172 nt from the 5′-end or 236 nt from the 3′-end of the 5′ UTR are removed. A truncated 5′ UTR with a calculated free energy of less than -272 kJ (-65 kcal/mol) is unable to support the synthesis in vitro of ODC protein. The short open reading frame (ORF) present in the 5′ UTR of rat ODC mRNA does not contribute to the observed inhibitory effect on translation efficiency in vitro. At low polyamine concentration the efficiency of translation in vitro of intact ODC mRNA is not relatively increased compared with that of an ODC mRNA having a truncated 5′ UTR or with that of control globin mRNA. From this we conclude that the well-documented negative feedback control of intracellular polyamines on ODC expression is not regulated by effects of polyamines on the secondary structure of the 5′ UTR of ODC mRNA.

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