In vitro RNA selection identifies RNA ligands that specifically bind to eukaryotic translation initiation factor 4B: the role of the RNA remotif.
Translation initiation factor elF-4B is an RNA-binding protein that promotes the association of the mRNA to the 40S ribosomal subunit. One of its better characterized features is the ability to stimulate the activity of the DEAD box RNA hilicase elF-4A. In addition to an RNA recognition motif (RRM) located near its amino-terimus, elF-4B contains an RNA-binding region in its carboxy-terminal half. The elF-4A helicase stimulatory activity resides in the carboxy-terminal half of elF-4B, and the RRM has little impact on this function. To better understand the role of the elF-4B RRM, it was of interest to identify its specific RNA target sequence. To this end, it vitro RNA selection/amplifications were performed using various portions of elF-4B. These experiments were designed to test the RNA recognition specificity of the two elF-4B regions implicated in RNA binding and to assess the influence of elF-4A on the RNA-binding specificity. The RRM was shown to bind with high affinity to an RNA stem-loop structure with conserved primary sequence elements. Discrete point mutations in an in vitro-selected RNA identified residues critical for RNA binding. Neither the carboxy-terminal RNA-interaction region, nor elF-4A, influenced the structure of the high-affinity RNA ligands selected by elF-4B, and elF-4A by itself did not select any specific RNA target. Previous studies have demonstrated an interaction of elF-4B with ribosomes, and it was suggested that this association is mediated through binding to ribosomal RNA. We show that the RRM of elF-4B interacts directly with 18S rRNA and this interaction is inhibited by an excess of the elF-4B in vitro-selected RNA. ElF-4B could bind simultaneously to two different RNA molecules, supporting a model whereby elF-4B promotes ribosome binding to the 5 untranslated region of a mRNA by bridging it to 18S rRNA.
Methot
N
N
Department of Biochemistry, McGill Cancer Centre, McGill University, Montreal, Quebec, Canada.
Pickett
G
G
Keene
J D
JD
Sonenberg
N
N
D001483:Base Sequence; D039642:Eukaryotic Initiation Factors; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010448:Peptide Initiation Factors / Q000378:metabolism*; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*
D039642:Eukaryotic Initiation Factors; D010448:Peptide Initiation Factors; D016601:RNA-Binding Proteins; C031799:eIF-4B; D012313:RNA