Open AccessDefining a novelciselement in the 3′-untranslated region of mammalian ribonucleotide reductase component R2 mRNA: role in transforming growth factor-β1induced mRNA stabilization
Author(s) -
Francis M. Amara,
Youhua Chen,
Jim A. Wright
Publication year - 1995
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/23.9.1461
Subject(s) - biology , ribonucleotide , ribonucleotide reductase , messenger rna , microbiology and biotechnology , untranslated region , au rich element , rna binding protein , rna , three prime untranslated region , biochemistry , gene , nucleotide , protein subunit
Ribonucleotide reductase R2 gene expression is elevated in BALB/c 3T3 fibroblasts treated with transforming growth factor beta 1. We investigated the possibility that the 3'-UTR of ribonucleotide reductase R2 mRNA contains regulatory information for TGF-beta 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 nucleotide (nt) cis element, 5'-GAGUUUGAG-3' site, which interacted specifically with a cytosolic protease sensitive factor to form a 75 kDa complex. The cis element protein binding activity was inducible and markedly up-regulated cross-link 4 h after TGF-beta 1 treatment of mouse BALB/c 3T3 cells. Other 3'-UTRs [IRE, GM-CSF, c-myc and homopolymer (U)] were poor competitors to the cis element with regard to forming the TGF-beta 1 dependent RNA-protein complex. However, the cis 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-beta 1 dependent RNA-protein band shifts as cell extract from BALB/c 3T3 mouse fibroblasts. Binding was completely prevented by several different mutations within the cis element, and by substitution mutagenesis, 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 cis to destabilize R2 mRNA in cells; and upon activation, a TGF-beta 1 responsive protein is induced and interacts with the 9 nt cis 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-beta 1-mediated effects.