Liu Y P, Nemeroff M, Yan Y P, Chen K Y
Department of Chemistry, Rutgers-State University of New Jersey 08855-0939, USA.
Biol Signals. 1997 May-Jun;6(3):166-74. doi: 10.1159/000109123.
Hypusine formation on the eukaryotic initiation factor 5A (eIF-5A) precursor represents a unique posttranslational modification that is ubiquitously present in eukaryotic cells and archaebacteria. Specific inhibition of deoxyhypusine synthase leads to growth arrest and cell death. The precise cellular function of eIF-5A and the physiological significance of hypusine modification are not clear. Although the methionyl-puromycin synthesis has been suggested to be the functional assay for eIF-5A activity in vitro, the role of eIF-5A in protein synthesis has not been established. Recent studies have suggested that eIF-5A may be the cellular target of the human immunodeficiency virus type 1 Rev and human T cell leukemia virus type 1 Rex proteins. Motif analysis suggested that eIF-5A resembles a bimodular RNA-binding protein in that it contains a stretch of basic amino acids clustered at the N-terminal region and a leucine-rich stretch at the C-terminal region. Using Rev target RNA, RRE, as a model, we tested the hypothesis that eIF-5A may be an RNA-binding protein. We found that both deoxyhypusine and hypusine-containing eIF-5A can bind to the 252-nt RRE RNA, as determined by a gel mobility shift assay. In contrast, the unmodified eIF-5A precursor cannot. Deoxyhypusine-containing eIF-5A, but not its precursor, could also cause supershift of the Rev stem-loop IIB RRE complex. Preliminary studies also indicated that eIF-5A can bind to RNA such as U6 snRNA and that deoxyhypusine modification appears to be required for the binding. The ability of eIF-5A to directly interact with RNA suggests that deoxyhypusine formation of eIF-5A may be related to its role in RNA processing and protein synthesis. Our study also suggests the possibility of using a gel mobility shift assay for eIF-5A-RNA binding as a functional assay for deoxyhypusine and hypusine formation.
真核生物起始因子5A（eIF-5A）前体上的hypusine形成代表一种独特的翻译后修饰，普遍存在于真核细胞和古细菌中。脱氧hypusine合酶的特异性抑制导致生长停滞和细胞死亡。eIF-5A的确切细胞功能以及hypusine修饰的生理意义尚不清楚。尽管甲硫氨酰-嘌呤霉素合成已被认为是体外eIF-5A活性的功能检测方法，但eIF-5A在蛋白质合成中的作用尚未确定。最近的研究表明，eIF-5A可能是人免疫缺陷病毒1型Rev和人T细胞白血病病毒1型Rex蛋白的细胞靶点。基序分析表明，eIF-5A类似于一种双模块RNA结合蛋白，因为它在N端区域含有一段聚集的碱性氨基酸，在C端区域含有一段富含亮氨酸的序列。以Rev靶RNA即RRE为模型，我们测试了eIF-5A可能是一种RNA结合蛋白的假设。我们发现，通过凝胶迁移率变动分析确定，含脱氧hypusine和含hypusine的eIF-5A都能与252 nt的RRE RNA结合。相比之下，未修饰的eIF-5A前体则不能。含脱氧hypusine的eIF-5A，而不是其前体，也能导致Rev茎环IIB-RRE复合物的超迁移。初步研究还表明，eIF-5A能与U6 snRNA等RNA结合，并且脱氧hypusine修饰似乎是结合所必需的。eIF-5A直接与RNA相互作用的能力表明，eIF-5A的脱氧hypusine形成可能与其在RNA加工和蛋白质合成中的作用有关。我们的研究还表明，有可能将eIF-5A-RNA结合的凝胶迁移率变动分析用作脱氧hypusine和hypusine形成的功能检测方法。
