A 20-AMINO-ACID AUTONOMOUS RNA-BINDING DOMAIN CONTAINED IN AN ENOYL-COA HYDRATASE

A+U-rich elements in the 3' untranslated region of mRNA species coding for lymphokines and early response genes play a pivotal role in the c ontrol of their rapid turnover. In a search for corresponding trans-ac ting factors, we have previously affinity-purified and cloned a human 32-kDa A+U-binding protein, termed AUH. AUH exhibited dual activities, namely A+U-specific RNA-binding and catalytic activity as enoyl-CoA h ydratase. In this report we map the RNA-binding site by analysis of a series of deletion and substitution recombinant proteins. Ultraviolet cross-linking experiments demonstrated that the deletion of a 20-amino -acid segment, Lys109-Ile128, abolished more than 80% of the relative RNA-binding activity. This segment conferred RNA-binding activity when fused to maltose binding protein. Binding of this fusion protein to A +U-rich RNA was significantly competed by an AUUUA cluster and poly(U) , followed by poly(G), but not by poly(A) nor poly(C). Furthermore, RN A binding of the fusion protein was competed by a synthetic peptide co rresponding to Lys109-Ile128. Circular dichroic measurement indicated formation of a specific complex between this peptide and poly(U) but n ot with poly(A). The identified 20 amino acids therefore constitute an automonous RNA-binding domain, distinct from the RNA-recognition moti fs of the family of ribonucleoproteins or NAD/RNA-binding sites in deh ydrogenases found in hitherto reported A+U-binding proteins. Replaceme nt of Arg125 in this motif with Glu reduced binding twofold, indicatin g this residue is integral to the binding function. Deletion of other parts of the protein did not impair RNA binding to any significant ext ent. By contrast, the hydratase activity of AUH required an intact thr ee-dimensional conformation, as most mutations downstream of Ser68 imp aired enzymatic activity.