High affinity RNA for mammalian initiation factor 4E interferes with mRNA-cap binding and inhibits translation

• Published in: RNA (Cambridge) 2005-01, Vol.11 (1), p.77-89
• Main Authors: Mochizuki, Kiyotaka, Oguro, Akihiro, Ohtsu, Takashi, Sonenberg, Nahum, Nakamura, Yoshikazu
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Animals; Base Sequence; Carrier Proteins - genetics; Carrier Proteins - metabolism; Eukaryotic Initiation Factor-4E - genetics; Eukaryotic Initiation Factor-4E - metabolism; Genetic Variation; Hepatitis C virus; In Vitro Techniques; Mice; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Nucleic Acid Conformation; Phosphoproteins - genetics; Phosphoproteins - metabolism; Protein Biosynthesis; Protein Conformation; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; RNA Cap Analogs - chemistry; RNA Cap Analogs - genetics; RNA Cap Analogs - metabolism; RNA Caps - chemistry; RNA Caps - genetics; RNA Caps - metabolism; Static Electricity
• ISSN: 1355-8382; 1469-9001
• DOI: 10.1261/rna.7108205

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.