Hypusine is required for a sequence-specific interaction of eukaryotic initiation factor 5A with postsystematic evolution of ligands by exponential enrichment RNA

Hypusine is formed through a spermidine-dependent posttranslational modific ation of eukaryotic initiation factor 5A (eIF-5A) at a specific lysine resi due. The reaction is catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase. eIF-5A is the only protein in eukaryotes and archaebacteria kn own to contain hypusine, Although both eIF-5A and deoxyhypusine synthase ar e essential genes for cell survival and proliferation, the precise biologic al function of eIF-5A is unclear. We have previously proposed that eIF-5A m ay function as a bimodular protein, capable of interacting with protein and nucleic acid (Liu, Y. P., Nemeroff, M., van, Y, P., and Chen, K, Y, (1997) Biol, Signals 6, 166-174), Here we used the method of systematic evolution of ligands by exponential enrichment (SELEX) to identify the sequence spec ificity of the potential eIF-5A RNA targets. The post-SELEX RNA obtained af ter 16 rounds of selection exhibited a significant increase in binding affi nity for eIF-5A with an apparent dissociation constant of 1 x 10(-7) M, The hypusine residue was found to be critical for this sequence-specific bindi ng. The post-SELEX RNAs shared a high sequence homology characterized by tw o conserved motifs, UAACCA and AAUGUCACAC. The consensus sequence was deter mined as AAAUGUCACAC by sequence alignment and binding studies. BLAST analy sis indicated that this sequence was present in >400 human expressed sequen ce tag sequences. The C terminus of eIF-5A contains a cold shock domain-lik e structure, similar to that present in cold shock protein A (CspA), Howeve r, unlike CspA, the binding of eIF-5A to either the post-SELEX RNA or the 5 '-untranslated region of CspA mRNA did not affect the sensitivity of these RNAs to ribonucleases. These data suggest that the physiological significan ce of eIF-5A-RNA interaction depends on hypusine and the core motif of the target RNA.