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Tethered-function analysis reveals that eIF4E can recruit ribosomes independent of its binding to the cap structure

Published online by Cambridge University Press:  07 February 2001

ENNIO DE GREGORIO
Affiliation:
Gene Expression Programme, European Molecular Biology Laboratory, D-69117 Heidelberg, Germany
JULIE BARON
Affiliation:
Gene Expression Programme, European Molecular Biology Laboratory, D-69117 Heidelberg, Germany
THOMAS PREISS
Affiliation:
Gene Expression Programme, European Molecular Biology Laboratory, D-69117 Heidelberg, Germany
MATTHIAS W. HENTZE
Affiliation:
Gene Expression Programme, European Molecular Biology Laboratory, D-69117 Heidelberg, Germany
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Abstract

The cap-binding complex eIF4F is involved in ribosome recruitment during the initiation phase of translation and is composed of three subunits: eIF4E, -4G, and -4A. The m7GpppN cap-binding subunit eIF4E binds the N-terminal region of eIF4G, which in turn contacts eIF4A through its central and C-terminal regions. We have previously shown, through a tethered-function approach in transfected HeLa cells, that the binding of eIF4G to an mRNA is sufficient to drive productive translation (De Gregorio et al., EMBO J, 1999, 18:4865–4874). Here we exploit this approach to assess which of the other subunits of eIF4F can exert this function. eIF4AI or mutant forms of eIF4E were fused to the RNA-binding domain of the λ phage antiterminator protein N to generate the chimeric proteins λ4A, λ4E-102 (abolished cap binding), and λ4E-73–102 (impaired binding to both, the cap and eIF4G). The fusion proteins were directed to a bicistronic reporter mRNA by means of interaction with a specific λ-N binding site (boxB) in the intercistronic space. We show that λ4E-102, but neither the double mutant λ4E-73–102 nor λ4A, suffices to promote translation of the downstream gene in this assay. Coimmunoprecipitation analyses confirmed that all λ-fusion proteins are capable of interacting with the appropriate endogenous eIF4F subunits. These results reveal that eIF4E, as well as eIF4G, can drive ribosome recruitment independent of a physical link to the cap structure. In spite of its interaction with endogenous eIF4G, λ4A does not display this property. eIF4A thus appears to supply an essential auxiliary function to eIF4F that may require its ability to cycle into and out of this complex.

Type
Research Article
Copyright
© 2001 RNA Society

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