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 regions. These elements are selectively recognized by AUF1, a component of a multisubunit complex that may participate in the initiation of mRNA decay. In this study, we have investigated the recognition of AREs by AUF1 in vitro using oligoribonucleotide substrates. Gel mobility shift assays demonstrated that U-rich RNA targets were specifically bound by AUF1, generating two distinct RNA-protein complexes in a concentration-dependent manner. Chemical cross-linking revealed the interaction of AUF1 dimers to form tetrameric structures involving protein-protein interactions in the presence of high affinity RNA targets. From these data, a model of AUF1 association with AREs involving sequential dimer binding was developed. Using fluorescent RNA substrates, binding parameters of AUF1 dimer-ARE and tetramer-ARE equilibria were evaluated in solution by fluorescence anisotropy measurements. Using two AUF1 deletion mutants, sequences C-terminal to the RNA recognition motifs are shown to contribute to the formation of the AUF1 tetramer.ARE complex but are not obligate for RNA binding activity. Kinetic studies demonstrated rapid turnover of AUF1.ARE complexes in solution, suggesting that these interactions are very dynamic in character. Taken together, these data support a model where ARE-dependent oligomerization of AUF1 may function to nucleate the formation of a trans-acting, RNA-destabilizing complex in vivo.
Wilson
G M
GM
Department of Microbiology Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1064, USA.
Sun
Y
Y
Lu
H
H
Brewer
G
G
D001483:Base Sequence; D001665:Binding Sites; D001704:Biopolymers; D019281:Dimerization; D005454:Fluorescence Polarization; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D006801:Humans; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D001704:Biopolymers; C000630986:HNRNPD protein, human; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012313:RNA