Assembly of AUF1 oligomers on U-rich RNA targets by sequential dimer association

Many labile mammalian mRNAs are targeted for rapid cytoplasmic turnover by the presence of A + U-rich elements (AREs) within their 3'-untranslated reg ions, These elements are selectively recognized by AUF1, a component of a m ultisubunit complex that may participate in the initiation of mRNA decay, I n this study, we have investigated the recognition of AREs by AUF1 in vitro using oligoribonucleotide substrates. Gel mobility shift assays demonstrat ed that U-rich RNA targets were specifically bound by AUF1, generating two distinct RNA-protein complexes in a concentration-dependent manner. Chemica l cross-linking revealed the interaction of AUF1 dimers to form tetrameric structures involving protein-protein interactions in the presence of high a ffinity RNA targets. From these data, a model of AUF1 association with AREs involving sequential dimer binding was developed. Using fluorescent RNA su bstrates, binding parameters of AUF1 dimer-ARE and tetramer-ARE equilibria were evaluated in solution by fluorescence anisotropy measurements. Using t wo AUF1 deletion mutants, sequences C-terminal to the RNA recognition motif s are shown to contribute to the formation of the AUF1 tetramer ARE complex but are not obligate for RNA binding activity. Kinetic studies demonstrate d rapid turnover of AUF1 ARE complexes in solution, suggesting that these i nteractions are very dynamic in character. Taken together, these data suppo rt a model where ARE-dependent oligomerization of AUF1 may function to nucl eate the formation of a trans-acting, RNA-destabilizing complex in vivo.