Sequence requirements for RNA binding by HuR and AUF1

The stability of RNAs bearing AU-rich elements in their 3'-UTRs, and thus the level of expression of their protein products, is regulated by interactions with cytoplasmic RNA-binding proteins. Binding by HuR generally leads to mRNA stabilization and increased protein production, whereas binding by AUF1 isoforms generally lead to rapid degradation of the mRNA and reduced protein production. The exact nature of the interplay between these and other RNA-binding proteins remains unclear, although recent studies have shown close interactions between them and even suggested competition between the two for binding to their cognate recognition sequences. Other recent reports have suggested that the sequences recognized by the two proteins are different. We therefore performed a detailed in vitro analysis of the binding site(s) for HuR and AUF1 present in androgen receptor mRNA to define their exact target sequences, and show that the same sequence is contacted by both proteins. Furthermore, we analysed a proposed HuR target within the 3'-UTR of MTA1 mRNA, and show that the contacted bases lie outside of the postulated motif and are a better match to a classical ARE than the postulated motif. The defining features of these HuR binding sites are their U-richness and single strandedness.