title: High affinity RNA for mammalian initiation factor 4E interferes with mRNA-cap binding and inhibits translation.
abstract: The eukaryotic translation initiation factor 4F (eIF4F) consists of three polypeptides (eIF4A, eIF4G, and eIF4E) and is responsible for recruiting ribosomes to mRNA. eIF4E recognizes the mRNA 5'-cap structure (m7GpppN) and plays a pivotal role in control of translation initiation, which is the rate-limiting step in translation. Overexpression of eIF4E has a dramatic effect on cell growth and leads to oncogenic transformation. Therefore, an inhibitory agent to eIF4E, if any, might serve as a novel therapeutic against malignancies that are caused by aberrant translational control. Along these lines, we developed two RNA aptamers, aptamer 1 and aptamer 2, with high affinity for mammalian eIF4E by in vitro RNA selection-amplification. Aptamer 1 inhibits the cap binding to eIF4E more efficiently than the cap analog m7GpppN or aptamer 2. Consistently, aptamer 1 inhibits specifically cap-dependent in vitro translation while it does not inhibit cap-independent HCV IRES-directed translation initiation. The interaction between eIF4E and eIF4E-binding protein 1 (4E-BP1), however, was not inhibited by aptamer 1. Aptamer 1 is composed of 86 nucleotides, and the high affinity to eIF4E is affected by deletions at both termini. Moreover, relatively large areas in the aptamer 1 fold are protected by eIF4E as determined by ribonuclease footprinting. These findings indicate that aptamers can achieve high affinity to a specific target protein via global conformational recognition. The genetic mutation and affinity study of variant eIF4E proteins suggests that aptamer 1 binds to eIF4E adjacent to the entrance of the cap-binding slot and blocks the cap-binding pocket, thereby inhibiting translation initiation.
authors.affiliation: Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Mochizuki
authors.forename: Akihiro
authors.initials: A
authors.lastname: Oguro
authors.forename: Takashi
authors.initials: T
authors.lastname: Ohtsu
authors.forename: Nahum
authors.initials: N
authors.lastname: Sonenberg
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Nakamura
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins; D039561:Eukaryotic Initiation Factor-4E / Q000235:genetics / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors; D014644:Genetic Variation; D066298:In Vitro Techniques; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012314:RNA Cap Analogs / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012315:RNA Caps / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C499527:Eif4ebp1 protein, mouse; D039561:Eukaryotic Initiation Factor-4E; D039642:Eukaryotic Initiation Factors; D010750:Phosphoproteins; D012314:RNA Cap Analogs; D012315:RNA Caps; D011994:Recombinant Proteins