Skip to main content
NCBI home page
RNA logoLink to RNA
. 1998 Jul;4(7):816–827. doi: 10.1017/s1355838298971990

Cis-acting elements are required for selenium regulation of glutathione peroxidase-1 mRNA levels.

S L Weiss 1, R A Sunde 1
PMCID: PMC1369661  PMID: 9671054

Abstract

Classical glutathione peroxidase (GPX1) mRNA levels can decrease to less than 10% in selenium (Se)-deficient rat liver. The cis-acting nucleic acid sequence requirements for Se regulation of GPX1 mRNA levels were studied by transfecting Chinese hamster ovary (CHO) cells with GPX1 DNA constructs in which specific regions of the GPX1 gene were mutated, deleted, or replaced by comparable regions from unregulated genes such as phospholipid hydroperoxide glutathione peroxidase (GPX4). For each construct, stable transfectants were pooled two weeks after transfection, divided into Se-deficient (2 nM Se) or Se-adequate (200 nM Se) medium, and grown for an additional four days. On day of harvest, Se-deficient GPX1 and GPX4 activities averaged 13 +/- 2% and 15 +/- 2% of Se adequate levels, confirming that cellular Se status was dramatically altered by Se supplementation. RNA was isolated from replicate plates of cells and transfected mRNA levels were specifically determined by RNase protection assay. Analysis of chimeric GPX1/GPX4 constructs showed that the GPX4 3'-UTR can completely replace the GPX1 3'-UTR in Se regulation of GPX1 mRNA. We did not find any GPX1 coding regions that could be replaced by the corresponding GPX4 coding regions without diminishing or eliminating Se regulation of the transfected GPX1 mRNA. Further analysis of the GPX1 coding region demonstrated that the GPX1 Sec codon (UGA) and the GPX1 intron sequences are required for full Se regulation of transfected GPX1 mRNA levels. Mutations that moved the GPX1 Sec codon to three different positions within the GPX1 coding region suggest that the mechanism for Se regulation of GPX1 mRNA requires a Sec codon within exon 1. Lastly, we found that addition of the GPX1 3'-UTR to beta-globin mRNA can convey significant Se regulation to beta-globin mRNA levels when a UGA codon is placed within exon 1. We conclude that Se regulation of GPX1 mRNA requires a functional selenocysteine insertion sequence (SECIS) in the 3'-UTR and a Sec codon followed by an intron.

Full Text

The Full Text of this article is available as a PDF (521.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ahern S. M., Miyata T., Sadler J. E. Regulation of human tissue factor expression by mRNA turnover. J Biol Chem. 1993 Jan 25;268(3):2154–2159. [PubMed] [Google Scholar]
  2. Belgrader P., Cheng J., Maquat L. E. Evidence to implicate translation by ribosomes in the mechanism by which nonsense codons reduce the nuclear level of human triosephosphate isomerase mRNA. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):482–486. doi: 10.1073/pnas.90.2.482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bermano G., Arthur J. R., Hesketh J. E. Role of the 3' untranslated region in the regulation of cytosolic glutathione peroxidase and phospholipid-hydroperoxide glutathione peroxidase gene expression by selenium supply. Biochem J. 1996 Dec 15;320(Pt 3):891–895. doi: 10.1042/bj3200891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bermano G., Nicol F., Dyer J. A., Sunde R. A., Beckett G. J., Arthur J. R., Hesketh J. E. Selenoprotein gene expression during selenium-repletion of selenium-deficient rats. Biol Trace Elem Res. 1996 Mar;51(3):211–223. doi: 10.1007/BF02784076. [DOI] [PubMed] [Google Scholar]
  5. Berry M. J., Banu L., Chen Y. Y., Mandel S. J., Kieffer J. D., Harney J. W., Larsen P. R. Recognition of UGA as a selenocysteine codon in type I deiodinase requires sequences in the 3' untranslated region. Nature. 1991 Sep 19;353(6341):273–276. doi: 10.1038/353273a0. [DOI] [PubMed] [Google Scholar]
  6. Berry M. J., Banu L., Harney J. W., Larsen P. R. Functional characterization of the eukaryotic SECIS elements which direct selenocysteine insertion at UGA codons. EMBO J. 1993 Aug;12(8):3315–3322. doi: 10.1002/j.1460-2075.1993.tb06001.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Binder R., Horowitz J. A., Basilion J. P., Koeller D. M., Klausner R. D., Harford J. B. Evidence that the pathway of transferrin receptor mRNA degradation involves an endonucleolytic cleavage within the 3' UTR and does not involve poly(A) tail shortening. EMBO J. 1994 Apr 15;13(8):1969–1980. doi: 10.1002/j.1460-2075.1994.tb06466.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carter M. S., Li S., Wilkinson M. F. A splicing-dependent regulatory mechanism that detects translation signals. EMBO J. 1996 Nov 1;15(21):5965–5975. [PMC free article] [PubMed] [Google Scholar]
  9. Casey J. L., Koeller D. M., Ramin V. C., Klausner R. D., Harford J. B. Iron regulation of transferrin receptor mRNA levels requires iron-responsive elements and a rapid turnover determinant in the 3' untranslated region of the mRNA. EMBO J. 1989 Dec 1;8(12):3693–3699. doi: 10.1002/j.1460-2075.1989.tb08544.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chambers I., Frampton J., Goldfarb P., Affara N., McBain W., Harrison P. R. The structure of the mouse glutathione peroxidase gene: the selenocysteine in the active site is encoded by the 'termination' codon, TGA. EMBO J. 1986 Jun;5(6):1221–1227. doi: 10.1002/j.1460-2075.1986.tb04350.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cheng J., Belgrader P., Zhou X., Maquat L. E. Introns are cis effectors of the nonsense-codon-mediated reduction in nuclear mRNA abundance. Mol Cell Biol. 1994 Sep;14(9):6317–6325. doi: 10.1128/mcb.14.9.6317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cheng J., Maquat L. E. Nonsense codons can reduce the abundance of nuclear mRNA without affecting the abundance of pre-mRNA or the half-life of cytoplasmic mRNA. Mol Cell Biol. 1993 Mar;13(3):1892–1902. doi: 10.1128/mcb.13.3.1892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Christensen M. J., Burgener K. W. Dietary selenium stabilizes glutathione peroxidase mRNA in rat liver. J Nutr. 1992 Aug;122(8):1620–1626. doi: 10.1093/jn/122.8.1620. [DOI] [PubMed] [Google Scholar]
  14. DePalo D., Kinlaw W. B., Zhao C., Engelberg-Kulka H., St Germain D. L. Effect of selenium deficiency on type I 5'-deiodinase. J Biol Chem. 1994 Jun 10;269(23):16223–16228. [PubMed] [Google Scholar]
  15. Hafeman D. G., Sunde R. A., Hoekstra W. G. Effect of dietary selenium on erythrocyte and liver glutathione peroxidase in the rat. J Nutr. 1974 May;104(5):580–587. doi: 10.1093/jn/104.5.580. [DOI] [PubMed] [Google Scholar]
  16. Haile D. J., Rouault T. A., Tang C. K., Chin J., Harford J. B., Klausner R. D. Reciprocal control of RNA-binding and aconitase activity in the regulation of the iron-responsive element binding protein: role of the iron-sulfur cluster. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7536–7540. doi: 10.1073/pnas.89.16.7536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hatfield D., Diamond A., Dudock B. Opal suppressor serine tRNAs from bovine liver form phosphoseryl-tRNA. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6215–6219. doi: 10.1073/pnas.79.20.6215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hill K. E., Lyons P. R., Burk R. F. Differential regulation of rat liver selenoprotein mRNAs in selenium deficiency. Biochem Biophys Res Commun. 1992 May 29;185(1):260–263. doi: 10.1016/s0006-291x(05)80984-2. [DOI] [PubMed] [Google Scholar]
  19. Ho Y. S., Howard A. J., Crapo J. D. Nucleotide sequence of a rat glutathione peroxidase cDNA. Nucleic Acids Res. 1988 Jun 10;16(11):5207–5207. doi: 10.1093/nar/16.11.5207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jacobson A., Peltz S. W. Interrelationships of the pathways of mRNA decay and translation in eukaryotic cells. Annu Rev Biochem. 1996;65:693–739. doi: 10.1146/annurev.bi.65.070196.003401. [DOI] [PubMed] [Google Scholar]
  21. Kaptain S., Downey W. E., Tang C., Philpott C., Haile D., Orloff D. G., Harford J. B., Rouault T. A., Klausner R. D. A regulated RNA binding protein also possesses aconitase activity. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10109–10113. doi: 10.1073/pnas.88.22.10109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Knudsen S., Brunak S. Kissing loops hide premature termination codons in pre-mRNA of selenoprotein genes and in genes containing programmed ribosomal frameshifts. RNA. 1997 Jul;3(7):697–701. [PMC free article] [PubMed] [Google Scholar]
  23. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  25. Lawrence R. A., Sunde R. A., Schwartz G. L., Hoekstra W. G. Glutathione peroxidase activity in rat lens and other tissues in relation to dietary selenium intake. Exp Eye Res. 1974 Jun;18(6):563–569. doi: 10.1016/0014-4835(74)90062-1. [DOI] [PubMed] [Google Scholar]
  26. Lei X. G., Evenson J. K., Thompson K. M., Sunde R. A. Glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are differentially regulated in rats by dietary selenium. J Nutr. 1995 Jun;125(6):1438–1446. doi: 10.1093/jn/125.6.1438. [DOI] [PubMed] [Google Scholar]
  27. Maquat L. E. When cells stop making sense: effects of nonsense codons on RNA metabolism in vertebrate cells. RNA. 1995 Jul;1(5):453–465. [PMC free article] [PubMed] [Google Scholar]
  28. Martin G. W., 3rd, Harney J. W., Berry M. J. Selenocysteine incorporation in eukaryotes: insights into mechanism and efficiency from sequence, structure, and spacing proximity studies of the type 1 deiodinase SECIS element. RNA. 1996 Feb;2(2):171–182. [PMC free article] [PubMed] [Google Scholar]
  29. Moscow J. A., Morrow C. S., He R., Mullenbach G. T., Cowan K. H. Structure and function of the 5'-flanking sequence of the human cytosolic selenium-dependent glutathione peroxidase gene (hgpx1). J Biol Chem. 1992 Mar 25;267(9):5949–5958. [PubMed] [Google Scholar]
  30. Pushpa-Rekha T. R., Burdsall A. L., Oleksa L. M., Chisolm G. M., Driscoll D. M. Rat phospholipid-hydroperoxide glutathione peroxidase. cDNA cloning and identification of multiple transcription and translation start sites. J Biol Chem. 1995 Nov 10;270(45):26993–26999. doi: 10.1074/jbc.270.45.26993. [DOI] [PubMed] [Google Scholar]
  31. Rhoads D. D., Roufa D. J. Emetine resistance of Chinese hamster cells: structures of wild-type and mutant ribosomal protein S14 mRNAs. Mol Cell Biol. 1985 Jul;5(7):1655–1659. doi: 10.1128/mcb.5.7.1655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Saedi M. S., Smith C. G., Frampton J., Chambers I., Harrison P. R., Sunde R. A. Effect of selenium status on mRNA levels for glutathione peroxidase in rat liver. Biochem Biophys Res Commun. 1988 Jun 16;153(2):855–861. doi: 10.1016/s0006-291x(88)81174-4. [DOI] [PubMed] [Google Scholar]
  33. Schuckelt R., Brigelius-Flohé R., Maiorino M., Roveri A., Reumkens J., Strassburger W., Ursini F., Wolf B., Flohé L. Phospholipid hydroperoxide glutathione peroxidase is a selenoenzyme distinct from the classical glutathione peroxidase as evident from cDNA and amino acid sequencing. Free Radic Res Commun. 1991;14(5-6):343–361. doi: 10.3109/10715769109093424. [DOI] [PubMed] [Google Scholar]
  34. Shen Q., Chu F. F., Newburger P. E. Sequences in the 3'-untranslated region of the human cellular glutathione peroxidase gene are necessary and sufficient for selenocysteine incorporation at the UGA codon. J Biol Chem. 1993 May 25;268(15):11463–11469. [PubMed] [Google Scholar]
  35. Stadtman T. C. Selenocysteine. Annu Rev Biochem. 1996;65:83–100. doi: 10.1146/annurev.bi.65.070196.000503. [DOI] [PubMed] [Google Scholar]
  36. Sunde R. A., Dyer J. A., Moran T. V., Evenson J. K., Sugimoto M. Phospholipid hydroperoxide glutathione peroxidase: full-length pig blastocyst cDNA sequence and regulation by selenium status. Biochem Biophys Res Commun. 1993 Jun 30;193(3):905–911. doi: 10.1006/bbrc.1993.1711. [DOI] [PubMed] [Google Scholar]
  37. Toyoda H., Himeno S., Imura N. Regulation of glutathione peroxidase mRNA level by dietary selenium manipulation. Biochim Biophys Acta. 1990 Jun 21;1049(2):213–215. doi: 10.1016/0167-4781(90)90042-z. [DOI] [PubMed] [Google Scholar]
  38. Weiss S. L., Evenson J. K., Thompson K. M., Sunde R. A. The selenium requirement for glutathione peroxidase mRNA level is half of the selenium requirement for glutathione peroxidase activity in female rats. J Nutr. 1996 Sep;126(9):2260–2267. doi: 10.1093/jn/126.9.2260. [DOI] [PubMed] [Google Scholar]
  39. Weiss S. L., Sunde R. A. Selenium regulation of classical glutathione peroxidase expression requires the 3' untranslated region in Chinese hamster ovary cells. J Nutr. 1997 Jul;127(7):1304–1310. doi: 10.1093/jn/127.7.1304. [DOI] [PubMed] [Google Scholar]
  40. Yoshimura S., Takekoshi S., Watanabe K., Fujii-Kuriyama Y. Determination of nucleotide sequence of cDNA coding rat glutathione peroxidase and diminished expression of the mRNA in selenium deficient rat liver. Biochem Biophys Res Commun. 1988 Aug 15;154(3):1024–1028. doi: 10.1016/0006-291x(88)90242-2. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES