Analysis of mRNA Recruitment to Ribosomes in Eukaryotic Cells

Thomas Preiss
Gene Expression Programme
Meyerhofstrasse 1
D-69117 Heidelberg Germany
telephone: +6221 387 139
fax: +6221 387 518
prestype: Platform
presenter: Thomas Preiss

Thomas Preiss, Julie Baron, Ennio De Gregorio, and Matthias W. Hentze
Gene Expression Programme, EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany

Nearly all eukaryotic mRNAs carry both a 5’ cap structure and a 3’ poly(A) tail as posttranscriptional modifications. The two modified mRNA termini are known to jointly and synergistically activate translation mediated by bridging interactions of eIF4E and PABP, with the translational adapter eIF4G, enabling a "circular" conformation of the mRNA. eIF4G also binds to eIF4A, an ATP-dependent RNA helicase, and through contacting the small ribosomal subunit-bound factor eIF3, is thought to effect it’s recruitment to the mRNA, a central step in translation initiation. In addition to these ‘core’ interactions, eIF4G also displays a growing number of interactions with other factors, which can modulate translation initiation or have the potential to link it to other aspects of the mRNA’s life cycle. We are investigating the functional consequences of interactions involving eIF4G using in vitro translation systems as well as cultured cells.

A tethered-function approach has been developed to study initiation factor function in transfected HeLa cells. The factor under investigation is expressed as a fusion to an RNA- binding domain, in the presence of a bicistronic reporter mRNA bearing a specific binding site for the fusion protein in the intercistronic space. Specific ribosome recruitment activity is then scored as translation of the downstream cistron. This assay allows the dissection of central steps in the early initiation pathway in the context of unperturbed cellular translation. We demonstrated that the central part of eIF4G, devoid of eIF4E- and PABP-interaction domains, functions as a "ribosome recruitment core". eIF4E, but not eIF4A, is also active in this assay, in a manner that does not require an interaction with the cap structure, but depends on binding to eIF4G.

Genetic and biochemical analyses in yeast (in collaboration with the labs of Iain Mattaj and Alan Sachs) demonstrated an interaction between eIF4G and the nuclear cap-binding complex CBC and that this interaction is antagonised by eIF4E. Furthermore, we found that CBC can stimulate translation in extracts containing an eIF4G protein deficient for eIF4E binding. These data suggest that eIF4E binding to the eIF4G-CBC complex on newly exported mRNA displaces CBC, thus facilitating an exchange of nuclear for cytoplasmic factors, and that the first round of translation on mRNA may occur via a different mechanism than subsequent rounds.

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