Characterization of proteins binding the 3′ regulatory region of the IL-3 gene in IL-3-dependent and autocrine-transformed hematopoietic cells

Abstract

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.