PHORBOL ESTER MODULATION OF A NOVEL CYTOPLASMIC PROTEIN-BINDING ACTIVITY AT THE 3'-UNTRANSLATED REGION OF MAMMALIAN RIBONUCLEOTIDE REDUCTASE R2 MESSENGER-RNA AND ROLE IN MESSAGE STABILITY

The R2 gene of ribonucleotide reductase is elevated in BALB/c 3T3 fibr oblasts treated with the tumor prometer, 12-O-tetradecanoylphorbol-13- acetate (TPA). TPA treatment increased the half-life of the R2 message by 3-fold, showing that TPA regulates R2 gene expression by a post-tr anscriptional mechanism(s). A 20-nucleotide (nt) TPA-responsive region was found within the R2 mRNA 3'-untranslated region (3'UTR), Ultravio let crosslinking detected a novel 45-kDa protein-R2 mRNA complex migra tion band that bound selectively to the 20-nt fragment and did not bin d to the 5'UTR or the coding region of the R2 message, or to the 3'UTR s of mRNAfrom several other genes, or to the homopolymer poly(A) se qu ence. The-45 KDa protein-R2 mRNA binding activity observed in unstimul ated cells was markedly down regulated after TPA treatment. Deletion o f a 20-nt region, containing the 20-nt sequence, from the 3'UTR caused stabilization of hybrid chloramphenicol acetyltransferase mRNA in the absence of TPA treatment. Furthermore, in vitro decay reaction mixtur es supplemented with the 20-nt sense RNA transcript resulted in stabil ization of R2 message. A model is presented of R2 message regulation i n which a vis-element within the 20-nt sequence of the 3'UTR interacts with a cytosolic protein to form a 45-kDa protein mRNA binding comple x. The TPA-induced alteration of R2 message stability is at least in p art due to the down regulation of the 45-kDa protein-mRNA binding acti vity which is linked to a reduction in the rate of R2 mRNA degradation .