A novel transforming growth factor-β1responsive cytoplasmic frans-acting factor binds selectively to the 3′-untranslated region of mammalian ribonucleotide reductase R2 mRNA: role in message stability

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Abstract
Ribonucleotide reductase is a highly regulated enzyme that provides the four deoxyribonucleotides required for DNA synthesis. Our studies showed that TGF-β1 treatment of BALB/c 3T3 mouse fibroblasts markedly elevated ribonucleotide reductase R2 mRNA levels, and also increased the half-life of R2 message by 4-fold from 1.5 h in untreated cells to 6 h in treated cells. We describe a novel 75 Kd sequence-specific cytoplasmic factor (p75) that binds selectively to a 83-nucleotide 3′-untranslated region of R2 mRNA and did not bind to the 5′UTR, the coding region of the R2 message or to the 3′UTRs of other mRNAs (from c-myc, GM-CSF and the iron responsive element from the transferrin receptor mRNA), or to the homopolymer poly(A) sequence. p75-RNA binding activity, which requires new protein synthesis, is not present in untreated cells, but is induced following TGF-β1 stimulation. The in vivo kinetics of appearance of p75 binding activity paralleled the accumulation of R2 mRNA. Insertion of the 3′-untranslated region into the chloramphenicol acetyltransferase (CAT) message confers TGF-β1 induced stability of RNA in stably transfected cells, while the same insert carrying a deletion of the 83-nucleotide fragment had little affect on RNA levels. Furthermore, in vitro decay reactions that contained the 83-nucleotide RNA or deletion of this fragment caused a significant decrease in TGF-β1 stabilization of R2 message. A model is presented of R2 message regulation in which TGF-β1 mediated stabilization of R2 message involves a specific interaction of a p75-frans-acting factor with a cis-element(s) stability determinant within the 83-nucleotide sequence which is linked to a reduction in the rate of R2 mRNA degradation.

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