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PMID: 9671054 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

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

RNA (New York, N.Y.) ·Vol. 4 ·No. 7 ·1998-07-00 ·Pages 816-27

Weiss SL, Sunde RA

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.

MeSH Terms
Animals CHO Cells Cricetinae Down-Regulation Gene Expression Regulation, Enzymologic Globins/biosynthesis,genetics Glutathione Peroxidase/biosynthesis,genetics Mice RNA, Messenger/biosynthesis Recombinant Fusion Proteins/biosynthesis,genetics Regulatory Sequences, Nucleic Acid Selenium/pharmacology Transfection
Chemicals
RNA, Messenger Recombinant Fusion Proteins Globins Glutathione Peroxidase Selenium
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Weiss S L
Department of Biochemistry, University of Missouri, Columbia 65211, USA.
Sunde R A
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Article Info
Journal
RNA (New York, N.Y.)
Abbr.
RNA
ISSN
1355-8382
DOI
10.1017/s1355838298971990
Published
1998-07-00
Pages
816-27
Language
English
Region
United States
NLM ID
9509184
PMCID
PMC1369661
Subset
IM
External Links
PubMed source DOI: 10.1017/s1355838298971990 PMC Full Text View journal details
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