Abstract
Mammalian ribonucleotide reductase is a highly regulated activity essential for DNA synthesis and repair. The 3'-untranslated region (3'-UTR) of mammalian ribonucleotide reductase R2 mRNA has been implicated in the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate-mediated stabilization of mouse BALB/c 3T3 R2 message. We investigated the possibility that the 3'-UTR contains regulatory information for R2 mRNA turnover. Using 3'-end-labeled RNA in gel shift and UV cross-linking analyses, we detected in the 3'-UTR a novel 9-nucleotide cis-element, 5'-UCGUGUGCU-3', which interacted with a widely distributed cellular cytosolic protease-sensitive factor(s) in a sequence-specific manner to form a 45-kDa R2 binding protein complex. The binding activity was redox-sensitive and down-regulated by 12-O-tetradecanoylphorbol-13-acetate and okadaic acid in a dose-dependent manner. Insertion of a 154-base pair fragment containing the cis-element led to markedly reduced accumulation of chloramphenicol acetyltransferase hybrid mRNA relative to the same insert carrying a series of G --> A mutations within this element that eliminated binding. We suggest that the 9-nucleotide region functions as a destabilizing element. These results provide a model for ribonucleotide reductase gene expression through a novel and specific mRNA cis-trans-interaction involving a phosphorylation signal pathway that leads to changes in the stability of R2 message.
Highlights
The regulation of mRNA stability has emerged as an important control mechanism of gene expression
We have recently shown that a 20-nt fragment (FokI-HgiA) in the 3Ј-UTR of R2 mRNA is involved in the TPA-mediated stabilization of R2 message [8]
Mammalian ribonucleotide reductase is composed of two dissimilar dimeric protein components, proteins R1 and R2, which are required to catalyze the direct reduction of ribonucleoside diphosphates to the corresponding deoxyribonucleotides, a rate-limiting step in the synthesis and repair of DNA [1, 2]
Summary
The regulation of mRNA stability has emerged as an important control mechanism of gene expression. The wild-type DNA fragment (R2 normal) containing 154 base pairs of the 3Ј-UTR of R2 mRNA (nt positions 1224 –1378), with the putative R2 binding protein complex (R2BP) cis-element, was generated by PCR with the plasmid pCD10 [8], harboring the R2 cDNA and the overlap extension primers, 5Ј-ACACACGTCGACGCTGACTTCTAAGTAACTC-3Ј and 5Ј-CGACACATCGATGCAGTCAGCACAGATCTACACAC-3Ј.
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