Characterization of the interaction between neuronal RNA-binding protein HuD and AU-rich RNA

Hu proteins have been shown to bind to AU-rich elements (AREs) in the 3'-untranslated region of unstable mRNAs. They can thereby inhibit the decay of labile transcripts by antagonizing destabilizing proteins that target these AU-rich sequences. Here we examine the sequence preferences of HuD to elucidate its possible role in counteracting mRNA decay. Using repeats of the prototype destabilizing sequence UU(AUUU)(n)AUU, we show that all three HuD RNA-binding domains participate in binding to AU-tracts that can be as short as 13 residues, depending on the position of the remaining As. Removal of the A residues, resulting in a poly(U)-tract, increased the affinity of HuD for RNA, suggesting that the presence of As in destabilizing elements might favor the recruitment of other proteins and/or prevent HuD from binding too tightly to AREs. In vitro selection experiments with randomized RNAs confirmed the preference of HuD for poly(U). RNA binding analysis of the related protein HuB showed a similar preference for poly(U). In contrast, tristetraprolin, an mRNA destabilizing protein, strongly prefers AU-rich RNA. Many labile mRNAs contain U-tracts in or near their AREs. Individual AREs may thus differentially affect mRNA half-life by recruiting a unique complement of stabilizing and destabilizing factors.