Abstract
In mammals, A+U-rich elements (AREs) are potent cis-acting determinants of rapid cytoplasmic mRNA turnover. Recognition of these sequences by AUF1 is associated with acceleration of mRNA decay, likely involving recruitment or assembly of multi-subunit trans-acting complexes. Previously, we demonstrated that AUF1 deletion mutants formed tetramers on U-rich RNA substrates by sequential addition of protein dimers (Wilson, G. M., Sun, Y., Lu, H., and Brewer, G. (1999) J. Biol. Chem. 274, 33374-33381). Here, we show that binding of the full-length p37 isoform of AUF1 to these RNAs proceeds via a similar mechanism, allowing delineation of equilibrium binding constants for both stages of tetramer assembly. However, association of AUF1 with the ARE from tumor necrosis factor (TNFalpha) mRNA was significantly inhibited by magnesium ions. Further fluorescence and hydrodynamic experiments indicated that Mg(2+) induced or stabilized a conformational change in the TNFalpha ARE. Based on the solution of parameters describing both the protein-RNA and Mg(2+)-RNA equilibria, we present a dynamic, global equilibrium binding model describing the relationship between Mg(2+) and AUF1 binding to the TNFalpha ARE. These studies provide the first evidence that some AREs may adopt higher order RNA structures that regulate their interaction with trans-acting factors and indicate that mRNA structural remodeling has the potential to modulate the turnover rates of some ARE-containing mRNAs.
Highlights
In mammals, A؉U-rich elements (AREs) are potent cis-acting determinants of rapid cytoplasmic mRNA turnover
Based on the solution of parameters describing both the protein-RNA and Mg2؉-RNA equilibria, we present a dynamic, global equilibrium binding model describing the relationship between Mg2؉ and AUF1 binding to the TNF␣ ARE
Association of Recombinant AUF1 Protein with the TNF␣ ARE Is Inhibited by Mg2ϩ—Previously, we demonstrated that a recombinant p37AUF1 deletion mutant lacking the C-terminal 29 amino acid residues (His6-p37AUF1-(1–257)) interacts with the core ARE sequence from TNF␣ mRNA, encoded by the RNA substrate Fl-TNF␣ ARE (Table I), by monitoring changes in the anisotropy of the fluorescein moiety of the RNA resulting from protein binding [25]
Summary
Based on the solution of parameters describing both the protein-RNA and Mg2؉-RNA equilibria, we present a dynamic, global equilibrium binding model describing the relationship between Mg2؉ and AUF1 binding to the TNF␣ ARE These studies provide the first evidence that some AREs may adopt higher order RNA structures that regulate their interaction with trans-acting factors and indicate that mRNA structural remodeling has the potential to modulate the turnover rates of some ARE-containing mRNAs. In eukaryotes, the cytoplasmic concentration of an mRNA is a critical determinant of its potential for translation and the production rate of the encoded gene product. Based on independent assessments of AUF1ARE and Mg2ϩ-ARE binding equilibria, we have constructed a model for inhibition of AUF1 binding and oligomerization to the TNF␣ ARE in the presence of Mg2ϩ To our knowledge, this represents the first indication that higher order RNA structures may regulate the association of trans-acting factors with an ARE
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