scholarly journals 3′-untranslated sequences mediate post-transcriptional regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA by 25-hydroxycholesterol

1995 ◽  
Vol 307 (1) ◽  
pp. 233-238 ◽  
Author(s):  
J W Choi ◽  
D M Peffley
Keyword(s):  
Protein Synthesis ◽  
Transcriptional Regulation ◽  
Untranslated Region ◽  
Simian Virus 40 ◽  
Beta Globin ◽  
Hmg Coa Reductase ◽  
Reductase Mrna ◽  
Coa Reductase ◽  
Post Transcriptional Regulation ◽  
Rna Levels

In an earlier study [Choi, Lundquist and Peffley (1993) Biochem. J. 296, 859-866], we determined that 25-hydroxycholesterol regulates 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA through a post-transcriptional mechanism that requires protein synthesis. To investigate whether 3′-untranslated sequences play a role in 25-hydroxycholesterol-mediated post-transcriptional control, we ligated approx. 1400 bp of the 3′-untranslated region of HMG-CoA reductase cDNA to the coding region of human beta-globin DNA. beta-Globin-3′-untranslated reductase fusion constructs were then transiently expressed in Chinese hamster ovary fibroblasts under conditions known to regulate reductase mRNA. There were no differences in beta-globin RNA levels in transfected cells incubated with or without lovastatin, a competitive inhibitor of reductase. However, in the presence of lovastatin and an oxysterol, 25-hydroxycholesterol, beta-globin RNA levels were decreased approx. 2-fold. Inhibition of protein synthesis with cycloheximide blocked the effects of 25-hydroxycholesterol on beta-globin RNA. Moreover, replacing the 3′-untranslated sequences with 1367 bp of the simian virus 40 enhancer region eliminated the regulatory effect of 25-hydroxycholesterol. Because the fusion construct has no sterol regulatory elements necessary for transcription, our results indicate that the change in beta-globin RNA occurred at a post-transcriptional level. In addition, we have shown that the 3′-untranslated region of HMG-CoA reductase cDNA imparted oxysterol-mediated post-transcriptional regulation to beta-globin RNA, an effect that required protein synthesis.

scholarly journals Inhibition of protein synthesis in baby-hamster kidney cells blocks oxysterol-mediated suppression of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA at a post-transcriptional level

1993 ◽  
Vol 296 (3) ◽  
pp. 859-866 ◽  
Author(s):  
J W Choi ◽  
E N Lundquist ◽  
D M Peffley
Keyword(s):  
Protein Synthesis ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Baby Hamster Kidney ◽  
Hmg Coa Reductase ◽  
Fetal Bovine ◽  
Reductase Mrna ◽  
Coa Reductase

The effects of the protein-synthesis inhibitor cycloheximide on 25-hydroxycholesterol-mediated suppression of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA levels were evaluated in the baby-hamster kidney cell line C100. Cells cultured in medium supplemented with delipidized fetal bovine serum and 25 microM lovastatin for 12-24 h had a 5-fold higher level of HMG-CoA reductase mRNA than cells grown in medium supplemented with non-delipidized fetal bovine serum (FBS). The higher level was due to increased transcription, as determined by run-on assays with isolated nuclei. Addition of 25-hydroxycholesterol to lovastatin-treated cells lowered HMG-CoA reductase mRNA levels within 4 h of treatment to those of cells grown in FBS-supplemented medium. This decrease was due in part to a decrease in gene transcription. Cycloheximide added in conjunction with 25-hydroxycholesterol to lovastatin-treated cells blocked the suppression of mRNA levels, but did not block oxysterol-mediated suppression of transcription. In addition, cycloheximide added to cells grown in FBS-supplemented medium rapidly increased mRNA levels by 10-fold relative to untreated cells, with no comparable increase in transcription. No comparable increase in either the mRNA level or rate of transcription for beta-actin was observed under such conditions. These results indicate that cycloheximide specifically stabilizes HMG-CoA reductase mRNA in the presence of oxysterols and suggests that continuous synthesis of a short lived protein regulator is required for oxysterol-mediated suppression of HMG-CoA reductase mRNA at a post-transcriptional level.

Post-transcriptional regulation of ULBP1 ligand for the activating immunoreceptor NKG2D involves 3′ untranslated region

2011 ◽  
Vol 72 (6) ◽  
pp. 470-478 ◽  
Author(s):  
Heike Himmelreich ◽  
Arina Mathys ◽  
Aleksandra Wodnar-Filipowicz ◽  
Christian P. Kalberer
Keyword(s):  
Transcriptional Regulation ◽  
Untranslated Region ◽  
Post Transcriptional Regulation

scholarly journals Loss of transcriptional activation of three sterol-regulated genes in mutant hamster cells.

1993 ◽  
Vol 13 (9) ◽  
pp. 5175-5185 ◽  
Author(s):  
M J Evans ◽  
J E Metherall
Keyword(s):  
Transcriptional Regulation ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Cho Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Wild Type ◽  
Hmg Coa Reductase ◽  
Low Levels ◽  
Coa Reductase

Cholesterol biosynthesis and uptake are controlled by a classic end product-feedback mechanism whereby elevated cellular sterol levels suppress transcription of the genes encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and the low-density lipoprotein receptor. The 5'-flanking region of each gene contains a common cis-acting element, designated the sterol regulatory element (SRE), that is required for transcriptional regulation. In this report, we describe mutant Chinese hamster ovary (CHO) cell lines that lack SRE-dependent transcription. Mutant cell lines were isolated on the basis of their ability to survive treatment with amphotericin B, a polyene antibiotic that kills cells by interacting with cholesterol in the plasma membrane. Four mutant lines (SRD-6A, -B, -C, and -D) were found to be cholesterol auxotrophs and demonstrated constitutively low levels of mRNA for all three sterol-regulated genes even under conditions of sterol deprivation. The mutant cell lines were found to be genetically recessive, and all four lines belonged to the same complementation group. When transfected with a plasmid containing a sterol-regulated promoter fused to a bacterial reporter gene, SRD-6B cells demonstrated constitutively low levels of transcription, in contrast to wild-type CHO cells, which increased transcription under conditions of sterol deprivation. Mutation of the SREs in this plasmid prior to transfection reduced the level of expression in wild-type CHO cells deprived of sterols to the level of expression found in SRD-6B cells. The defect in SRD-6 cells is limited to transcriptional regulation, since posttranscriptional mechanisms of sterol-mediated regulation were intact: the cells retained the ability to posttranscriptionally suppress HMG-CoA reductase activity and to stimulate acyl-CoA:cholesterol acyltransferase activity. These results suggest that SRD-6 cells lack a factor required for SRE-dependent transcriptional activation. We contrast these cells with a previously isolated oxysterol-resistant cell line (SRD-2) that lacks a factor required for SRE-dependent transcriptional suppression and propose a model for the role of these genetically defined factors in sterol-mediated transcriptional regulation.

scholarly journals The 3′-untranslated region of the mouse cholesterol 7α-hydroxylase mRNA contains elements responsive to post-transcriptional regulation by bile acids

1997 ◽  
Vol 328 (2) ◽  
pp. 393-399 ◽  
Author(s):  
B. Luis AGELLON ◽  
K. Sukhinder CHEEMA
Keyword(s):  
Transcriptional Regulation ◽  
Bile Acids ◽  
Untranslated Region ◽  
Transgene Expression ◽  
Hepatoma Cells ◽  
Early Gene ◽  
Transcriptional Level ◽  
Acid Uptake ◽  
Post Transcriptional Regulation ◽  
Conjugated Bile Acids

To investigate the importance of the 3ʹ-untranslated region (UTR) of the mouse cholesterol 7α-hydroxylase (cyp7) mRNA in post-transcriptional regulation of expression of the cyp7 gene, chimaeric genes encoding mRNA containing the structural sequence of chloramphenicol acetyltransferase (CAT) linked to either the 3ʹ-UTR of the mouse cyp7 mRNA or the SV40 early gene mRNA were constructed. The human cytomegalovirus (CMV) promoter was used to drive the expression of all the chimaeric genes. Thus the transgenes had identical sequences in the promoter, the regions encoding the 5ʹ-UTR and translated sequence but differed in the region encoding the 3ʹ-UTR of their respective mRNA species. The transgene containing the entire cyp7 3ʹ-UTR (designated CMV.CAT.CYP7) gave rise to CAT activity in transfected hepatoma cells that was one-quarter of that obtained in cells transfected with the transgene containing the SV40 3ʹ-UTR (designated CMV.CAT.SV40). The 3ʹ-UTR of the cyp7 mRNA contains sequences resembling AU-rich elements (AREs). Deleting eight of nine putative AREs from the CYP7 3ʹ-UTR sequence increased the CAT activity to a level greater than that observed for CMV.CAT.SV40, whereas deletion of the intron region had no effect. These results show that the AREs of the 3ʹ-UTR of the cyp7 mRNA decrease transgene expression. Bile acids are known to repress the expression of the cyp7 gene. To test whether the 3ʹ-UTR of the cyp7 mRNA has a role in this process, the expression of the chimaeric genes was evaluated in hepatoma cells competent for bile acid uptake. Conjugated bile acids, but not unconjugated bile acids, further decreased the expression of the CMV.CAT.CYP7 transgene. The same bile acids had no effect on the expression of the CMV.CAT.SV40 transgene. Deletion of the intron from the cyp7 sequence did not alter the CAT activity compared with the parental plasmid, and also did not alter the sensitivity of the transgene to the conjugated bile acids. Deletion of the AREs from the cyp7 3ʹ-UTR, which increased the expression of the transgene, did not abolish the sensitivity of the transgene to repression by conjugated bile acids. Thus the 3ʹ-UTR of the mouse cyp7 mRNA also contains elements that facilitate the further repression of transgene expression in the presence of conjugated bile acids. The results indicate that the 3ʹ-UTR of the mouse cyp7 mRNA contains information specifying regulation at the post-transcriptional level.

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