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.
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.
Herrick
D J
DJ
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Ross
J
J
D016475:3T3 Cells; D000818:Animals; D002453:Cell Cycle; D003609:Dactinomycin / Q000494:pharmacology; D005786:Gene Expression Regulation; D016259:Genes, myc; D005914:Globins / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D010443:Peptide Chain Termination, Translational; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D014162:Transfection
D012333:RNA, Messenger; D003609:Dactinomycin; D005914:Globins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases