Posttranscriptional regulation of protein expression in human epithelial carcinoma cells by adenine-uridine-rich elements in the 3'-untranslated region of tumor necrosis factor-alpha messenger RNA.
Eukaryotic mRNAs contain 3'-untranslated regions (UTR) that are involved in posttranscriptional control of gene expression. AU-rich octanucleotide repeats, UUAUUUAU, present in the 3'-UTR of mature lymphokine and other cytokine transcripts, have been implicated in the regulation of mRNA stability and translational efficiency. For example, previous evidence suggests that the AU-rich element (ARE) present in the 3'-UTR of murine tumor necrosis factor-alpha (TNF-alpha) can affect the posttranscriptional regulation of murine TNF-alpha gene expression in hematopoietic cells. Although cytokines are produced in epithelial cells, little is known about the regulation of TNF-alpha and other cytokine gene expression by 3'-UTR elements in human malignant epithelial cells. To better understand the function of the 3'-UTR of the human TNF-alpha gene in the regulation of TNF-alpha protein production in human epithelial cancer cells, a series of luciferase reporter constructs with portions of the 3'-UTR of human TNF-alpha was transfected into human breast carcinoma cell lines ZR-75-1 and ZR-75-1R (which overexpresses TNF-alpha). The 3'-UTR of TNF-alpha markedly suppressed luciferase activity in both cell lines, and the suppression of activity was reversed by deletion of the AU-rich sequences. This suppression was quantitative, with six repeats causing more inhibition than two repeats. Increased levels of luciferase activity were observed 3 h after TNF-alpha stimulation in ZR-75-1 cells transfected by constructs containing AU-rich repeats. In addition, cytoplasmic extracts from both cell lines were assayed for factors that bind to the 3'-UTR of human TNF-alpha mRNA. RNA-protein binding activities were found in both cell lines. Competition studies showed that these proteins specifically bound to AU-rich repeats present in the 3'-UTR of TNF-alpha. No binding activity was observed when the AU-rich repeats were deleted. TNF-alpha exposure markedly increased activity of several RNA-binding proteins, especially a novel Mr 50,000-55,000 RNA-binding protein. The binding activity in untreated ZR-75-1R was higher than that in untreated ZR-75-1 cells, suggesting that the level of RNA-protein binding correlates with the expression level of TNF-alpha in human epithelial cancer cells and that the RNA-binding proteins may control expression of TNF-alpha in ZR-75-1 cells. We conclude that the AU-rich repeats in the 3'-UTR of human TNF-alpha mRNA may regulate gene expression in human epithelial cancer cells by binding to AU sequence-specific proteins, including a previously undescribed Mr 50,000-55,000 protein not observed in hematopoietic cells.
Wang
E
E
Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Ma
W J
WJ
Aghajanian
C
C
Spriggs
D R
DR
D000818:Animals; D001483:Base Sequence; D001943:Breast Neoplasms; D016207:Cytokines / Q000096:biosynthesis*; D004847:Epithelial Cells; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D017930:Genes, Reporter; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis; D051379:Mice; D009843:Oligoribonucleotides / Q000494:pharmacology*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
D016207:Cytokines; D009843:Oligoribonucleotides; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D008156:Luciferases