Hypusine Is Required for a Sequence-specific Interaction of Eukaryotic Initiation Factor 5A with Postsystematic Evolution of Ligands by Exponential Enrichment RNA

Abstract

Hypusine is formed through a spermidine-dependent posttranslational modification of eukaryotic initiation factor 5A (eIF-5A) at a specific lysine residue. The reaction is catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase. eIF-5A is the only protein in eukaryotes and archaebacteria known to contain hypusine. Although both eIF-5A and deoxyhypusine synthase are essential genes for cell survival and proliferation, the precise biological function of eIF-5A is unclear. We have previously proposed that eIF-5A may function as a bimodular protein, capable of interacting with protein and nucleic acid (Liu, Y. P., Nemeroff, M., Yan, 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 specificity of the potential eIF-5A RNA targets. The post-SELEX RNA obtained after 16 rounds of selection exhibited a significant increase in binding affinity for eIF-5A with an apparent dissociation constant of 1 ؋ 10 ؊7 M. The hypusine residue was found to be critical for this sequence-specific binding. The post-SELEX RNAs shared a high sequence homology characterized by two conserved motifs, UAACCA and AAUGUCACAC. The consensus sequence was determined as AAAUGUCA-CAC by sequence alignment and binding studies. BLAST analysis indicated that this sequence was present in >400 human expressed sequence tag sequences. The C terminus of eIF-5A contains a cold shock domain-like structure, similar to that present in cold shock protein A (CspA). However, 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 significance of eIF-5A-RNA interaction depends on hypusine and the core motif of the target RNA.Eukaryotic initiation factor 5A (eIF-5A), 1 ubiquitously present in eukaryotes and archaebacteria, but not in eubacteria, is the only protein known to contain a hypusine residue (for review, see Refs. 1-3). Hypusine is formed in two steps: (i) deoxyhypusine synthase catalyzes the transfer of a 4-aminobutyl moiety from spermidine to a specific lysine residue to form a deoxyhypusine residue, N ⑀ -(4-aminobutyl)lysine; and (ii) deoxyhypusine hydroxylase catalyzes the hydroxylation of the deoxyhypusine residue to form hypusine (N ⑀ -(4-amino-2-hydroxybutyl)lysine). The fact that nature has committed two enzymes to produce one hypusine residue on a single protein underscores the importance of this posttranslational modification. Hypusine formation is tightly coupled to cell proliferation and is essential for cell survival (1-3). Disruption of either the eIF-5A or deoxyhypusine synthase gene in yeast leads to a lethal phenotype (4 -6). Inhibition of deoxyhypusine synthase in mammalian cells causes growth arrest (7-9), cell death (10), or tumor differentiation (9). In addition, hypusine formation activity exhibits a marked increase in virally transformed cells (11) but ...

Destination-Selective Long-Distance Movement of Phloem Proteins

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Inhibition of Deoxyhypusine Synthase Enhances Islet β Cell Function and Survival in the Setting of Endoplasmic Reticulum Stress and Type 2 Diabetes

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