The 3'-untranslated region of apolipoprotein II mRNA contains two independent domains that bind distinct cytosolic factors

Abstract

The 3'-untranslated region of apolipoprotein II (apoII) mRNA contains target sites for mRNA breakdown (Binder, R., Hwang, S.-P. L., Ratnasabapathy, R., and Williams, D. L. (1989) J. Biol. Chem. 264, 16910-16918). Degradation occurs via endonucleolytic cleavage at 5'-AAU-3'/5'-UAA-3' elements in single-stranded loop domains of the 3'-untranslated region. Degradation target sites occur in two clusters that are localized within two larger domains of secondary structure. In this study, gel shift and label transfer assays were used to identify liver cytosolic factors that recognize the 3'-untranslated region of apoII mRNA. The results show preferential binding of cytosolic factors to the 3'-untranslated region as compared to the coding region. UV cross-linking experiments confirmed that cytosolic factors labeled by the 3'-untranslated region are a subset of proteins labeled by the entire mRNA. Two distinct binding domains were identified within the 3'-untranslated region. The upstream domain encompassing nucleotides 400-547 extends from the translation stop codon through the complex stem-loop D structure described previously. This domain labeled primarily a 34-kDa protein in UV cross-linking experiments. The downstream binding domain encompassing nucleotides 568-643 includes another region of secondary structure and terminates within the universal polyadenylation signal. The downstream domain labeled primarily a 60-kDa protein in UV cross-linking experiments. The upstream and downstream binding domains did not compete with each other in gel shift or cross-linking experiments. These results indicate that the 3'-untranslated region can form two independent messenger ribonucleoprotein complexes localized to domains that include target sites for apoII mRNA degradation. We speculate that these messenger ribonucleoprotein complexes may play a role in the degradation of apoII mRNA or in the regulation of this process.