A fibroblast protein binds the 3'-untranslated region of pro-alpha 1(I) collagen mRNA.
Post-transcriptional regulation of the expression of the pro alpha 1(I) chain of type I collagen (COL1A1) was studied by analysing cytoplasmic RNA-binding proteins and by transient transfections with collagen minigene plasmids. In this paper we present evidence for a factor from NIH 3T3 cells and human skin fibroblasts that interacts with the conserved 3'-untranslated region (UTR) of the shorter 4.8 kb mRNA species of the COL1A1 gene. The specificity of the interaction was confirmed by using (i) unlabelled specific and non-specific competitor RNAs and (ii) oligodeoxyribonucleotides annealed to the probe or used as single-stranded competitors. An antisense oligonucleotide annealed to the RNA probe near its 3'-terminus [20-42 nucleotides upstream of the first polyadenylation signal of the alpha 1(I) collagen mRNA] inhibited the binding, whereas other sense or antisense oligonucleotides had no effect on the interaction. The binding was sensitive to alkylation of free SH groups 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 alpha 1-RBF67 (type I collagen alpha 1 chain RNA-binding factor). Dexamethasone treatment of fibroblasts, which is known to accelerate the turnover of COL1A1 mRNA, decreased the alpha 1-RBF67 activity markedly as evaluated by gel-retardation and u.v. cross-linking assays. Transient transfections with plasmids carrying the alpha 1(I) collagen promoter and 3'-UTR sequences demonstrated that the 3'-UTR participates in the response to dexamethasone. Thus the loss of alpha 1-RBF67 activity might be associated with decreased alpha 1(I) collagen mRNA levels after dexamethasone treatment.
Määttä
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A
Department of Medical Biochemistry, University of Turku, Finland.
Penttinen
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RP
D016475:3T3 Cells / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D003907:Dexamethasone / Q000494:pharmacology; D005095:Exoribonucleases / Q000378:metabolism; D005347:Fibroblasts / Q000737:chemistry*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense; D011347:Procollagen / Q000235:genetics*; D015347:RNA Probes; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014162:Transfection; D014466:Ultraviolet Rays
D016376:Oligonucleotides, Antisense; D011347:Procollagen; D015347:RNA Probes; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D003907:Dexamethasone; D005095:Exoribonucleases; C042081:exoribonuclease T