Characterization of proteins binding the 3' regulatory region of the IL-3 gene in IL-3-dependent and autocrine-transformed hematopoietic cells.
Previously we documented the prolongation of the IL-3 mRNA half-life in an autocrine-transformed cell line. This cell line has an intracisternal type A particle transposition in the IL-3 mRNA 3' untranslated region which displaced four out of six AUUUA motifs involved in IL-3 mRNA destabilization. In this study, the proteins binding to the IL-3 mRNA AU-rich elements (ARE) were examined. Specific protein binding was detected to the wild-type IL-3 ARE region which contained 6 AUUUA motifs (AU6). In contrast, no binding was detected to the mutated IL-3 ARE region which contained only two AUUUA motifs (AU2). Proteins with apparent molecular weights of 36, 40, 43, 46, 55, 57, 68 and 95 kDa were bound to AU6 motif. The hnRNP C and AUF-1 (hnRNP D) proteins were determined to be two of the IL-3 ARE binding proteins. Incubation of protein extracts with antibodies to hnRNP C and AUF-1 significantly decreased the protein binding to the IL-3 ARE. Treatment of IL-3 dependent cells with calcium ionophores eliminated the proteins binding to the ARE in wild-type IL-3-dependent FL5.12 cells and also resulted in the accumulation of IL-3 mRNA transcripts with a long half-life. These results indicated that there was a specific complex which bound the IL-3 mRNA 3' ARE. Mutations which truncate the IL-3 ARE eliminate the ability of proteins to bind this regulatory region and can result in autocrine transformation due to the presence of IL-3 mRNA transcripts with a long half-life.
Wang
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Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC 27858, USA.
Hoyle
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PE
McCubrey
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JA
D000225:Adenine / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D007377:Interleukin-3 / Q000096:biosynthesis / Q000235:genetics*; D007476:Ionophores / Q000494:pharmacology; D051379:Mice; D015347:RNA Probes; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D012261:Ribonucleoproteins / Q000378:metabolism; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014170:Transformation, Genetic; D014498:Uracil / Q000378:metabolism
D007377:Interleukin-3; D007476:Ionophores; D015347:RNA Probes; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D000001:Calcimycin; D014498:Uracil; D000225:Adenine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium