HuD RNA recognition motifs play distinct roles in the formation of a stable complex with AU-rich RNA

Human neuron-specific RNA-binding protein HuD belongs to the family of Hu p roteins and consists of two N-terminal RNA recognition motifs (RRM1 and -2) , a hinge region, and a C-terminal RRM (RRM3). Hu proteins can bind to AU-r ich elements in the 3' untranslated regions of unstable mRNAs, causing the stabilization of certain transcripts. We have studied the interaction betwe en HuD and prototype mRNA instability elements of the sequence UU(AUUU)(n)A UU using equilibrium methods and real-time kinetics (surface plasmon resona nce using a BIACORE). We show that a single molecule of HuD requires at lea st three AUUU repeats to bind tightly to the RNA. Deletion of RRM1 reduced the K-d by 2 orders of magnitude and caused a decrease in the association r ate and a strong increase in the dissociation rate of the RNA-protein compl ex, as expected when a critical RNA-binding domain is removed. In contrast, deletion of either RRM2 or -3, which only moderately reduced the affinity, caused marked increases in the association and dissociation rates. The slo wer binding and stabilization of the complex observed in the presence of al l three RRMs suggest that a change in the tertiary structure occurs during binding. The individual RRMs bind poorly to the RNA (RRM1 binds with microm olar affinity, while the affinities of RRM2 and -3 are in the millimolar ra nge). However, the combination of RRM1 and either RRM2 or RRM3 in the conte xt of the protein allows binding with a nanomolar affinity. Thus, the three RRMs appear to cooperate not only to increase the affinity of the interact ion but also to stabilize the formed complex. Kinetic effects, similar to t hose described here, could play a role in RNA binding by many multi-RRM pro teins and may influence the competition between proteins for RNA-binding si tes and the ability of RNA-bound proteins to be transported intracellularly .