A FIBROBLAST PROTEIN BINDS THE 3'-UNTRANSLATED REGION OF PRO-ALPHA-1(I) COLLAGEN MESSENGER-RNA

Post-transcriptional regulation of the expression of the proalpha1(I) chain of type I collagen (COL1A1) was studied by analysing cytoplasmic RNA-binding proteins and by transient transfections with collagen min igene plasmids. In this paper we present evidence for a factor from NI H 3T3 cells and human skin fibroblasts that interacts with the conserv ed 3'-untranslated region (UTR) of the shorter 4.8 kb mRNA species of the COL1A1 gene. The specificity of the interaction was confirmed by u sing (i) unlabelled specific and non-specific competitor RNAs and (ii) oligodeoxyribonucleotides annealed to the probe or used as single-str anded competitors. An antisense oligonucleotide annealed to the RNA pr obe near its 3'-terminus [20-42 nucleotides upstream of the first poly adenylation signal of the alpha1(I) collagen mRNA] inhibited the bindi ng, whereas other sense or antisense oligonucleotides had no effect on the interaction. The binding was sensitive to alkylation of free SH g roups but not to phosphatase treatment of the extracts. In u.v. cross- linking analysis this factor migrated as a single polypeptide chain of about 67 kDa, and was named alpha1-RBF67 (type I collagen alpha1 chai n RNA-binding factor). Dexamethasone treatment of fibroblasts, which i s known to accelerate the turnover of COL1A1 mRNA, decreased the alpha 1-RBF67 activity markedly as evaluated by gel-retardation and u.v. cro ss-linking assays. Transient transfections with plasmids carrying the alpha1(I) collagen promoter and 3'-UTR sequences demonstrated that the 3'-UTR participates in the response to dexamethasone. Thus the loss o f alpha1-RBF67 activity might be associated with decreased alpha1(I) c ollagen mRNA levels after dexamethasone treatment.