Defining a novelciselement in the 3′-untranslated region of mammalian ribonucleotide reductase component R2 mRNA: role in transforming growth factor-β1induced mRNA stabilization

Abstract

Ribonucleotide reductase R2 gene expression is elevated In BALB/c 3T3 flbroblasts treated with transforming growth factor β ¹ We investigated the possibility that the 3′-UTR of ribonucleotide reductase R2 mRNA contains regulatory Information for TGF-β ₁ Induced message stability. Using end-labeled RNA fragments In gel shift assays and UV cross-linking analyses, we detected in the 3′-UTR a novel 9 nucleo-tide (nt) cls element, 5′ -GAGUUUGAG-3′ site, which Interacted specifically with a cytosolic protease sensitive factor to form a 75 kDa complex. The els element protein binding activity was induclble and markedly up-regulated cross-link 4 h after TGF-β ₁ treatment of mouse BALB/c 3T3 cells. Other 3′-UTRs [IRE, GM-CSF, c-myc and homopolymer (U)] were poor competitors to the cls element with regard to forming the TGF-β ₁ dependent RNA-proteln complex. However, the cls element effectively competed out the formation of the R2 3′-UTR protein complex. Cytosolic extracts from a variety of mammalian cell lines (monkey Cos7, several mouse fibrosarcomas and human HeLa S3) demonstrated similar TGF-β ₁ dependent RNA-protein band shifts as cell extract from BALB/c 3T3 mouse fibro-blasts. Binding was completely prevented by several different mutations within the cls element, and by substitution mutagenesls, we were able to predict the consensus sequences, 5′-GAGUUUNNN-3′ and 5′-NNNUUUGAG-3′ for optimal protein binding. These results support a model in which the 9 nt region functions In cls to destabilize R2 mRNA in cells; and upon activation, a TGF-β ₁ responsive protein is induced and interacts with the 9 nt cls element in a mechanism that leads to stabilization of the mRNA. This appears to be the first example of a mRNA binding site that is Involved In TGF-β ₁ -mediated effects.