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 i n an autocrine-transformed cell line. This cell line has an intraciste rnal type A particle transposition in the IL-3 mRNA 3' untranslated re gion which displaced four out of six AUUUA motifs involved in IL-3 mRN A destabilization. In this study, the proteins binding to the IL-3 mRN A AU-rich elements (ARE) were examined. Specific protein binding was d etected to the wildtype IL-3 ARE region which contained 6 AUUUA motifs (AU(6)), In contrast, no binding was detected to the mutated IL-3 ARE region which contained only two AUUUA motifs (AU(2)). Proteins with a pparent molecular weights of 36, 40, 43, 46, 55, 57, 68 and 95 kDa wer e bound to AU(6) motif. The hnRNP C and AUF-1 (hnRNP D) proteins were determined to be two of the IL-3 ARE binding proteins. Incubation of p rotein extracts with antibodies to hnRNP C and AUF-1 significantly dec reased the protein binding to the IL-3 ARE. Treatment of IL-3 dependen t 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 re sults indicated that there was a specific complex which bound the IL 3 mRNA 3' ARE, Mutations which truncate the IL-3 ARE eliminate the abil ity of proteins to bind this regulatory region and can result in autoc rine transformation due to the presence of IL-3 mRNA transcripts with a long half-life.