Identification of mRNA that binds to eukaryotic initiation factor 5A by affinity co-purification and differential display.
Eukaryotic initiation factor 5A (eIF-5A) is the only protein in nature that contains hypusine, an unusual amino acid formed post-translationally by deoxyhypusine synthase and deoxyhypusine hydroxylase. Genetic and pharmacological evidence suggests that eIF-5A is essential for cell survival and proliferation. However, the precise function and interacting partners of eIF-5A remain unclear. We have shown previously that eIF-5A can bind to RRE (Rev-response element) and U6 RNA in vitro. Using SELEX (systematic evolution of ligands by exponential enrichment), we have also shown that eIF-5A is capable of binding to RNA in a sequence-specific manner [Xu and Chen (2001) J. Biol. Chem. 276, 2555-2561]. In the present paper, we show that the identification of mRNA species that bind to eIF-5A can be achieved by affinity co-purification and PCR differential display. Using this approach with three sets of anchoring and arbitrary primers, we have found 20 RNA sequences that co-purified specifically with eIF-5A. Five of them contained AAAUGU, the putative eIF-5A-interacting element that we identified previously using the SELEX method. Direct binding of the cloned RNA to eIF-5A could be demonstrated by electrophoretic mobility-shift assay. BLAST analysis revealed that the eIF-5A-interacting RNAs encode proteins such as ribosomal L35a, plasminogen activation inhibitor mRNA-binding protein, NADH dehydrogenase subunit and ADP-ribose pyrophosphatase. Some, however, encode hypothetical proteins. All the cloned RNAs have the potential to form extensive stem-loop structures.
Department of Chemistry and Chemical Biology, Rutgers The State University of New Jersey, 610 Taylor Road, Piscataway, NJ 08854-8087, USA.
Aiguo
A
Xu
David Li-En
DL
Jao
Kuang Yu
KY
Chen
D001483:Base Sequence; D001665:Binding Sites; D002470:Cell Survival; D002846:Chromatography, Affinity; D019295:Computational Biology; D020869:Gene Expression Profiling; D006367:HeLa Cells; D006801:Humans; D009690:Nucleic Acid Conformation; D010448:Peptide Initiation Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection; D000097574:Eukaryotic Translation Initiation Factor 5A
D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000097574:Eukaryotic Translation Initiation Factor 5A