Differential Cleavage of eIF4GI and eIF4GII in Mammalian Cells: EFFECTS ON TRANSLATION

Alfredo Castelló,Enrique Alvarez,Luis Carrasco

doi:10.1074/jbc.m604340200

Abstract

Two isoforms of the translation initiation factor eIF4G, eIF4GI and eIF4GII, have been described in eukaryotic cells. The exact function of each isoform during the initiation of protein synthesis is still under investigation. We have developed an efficient and reliable method of expressing poliovirus 2Apro, which differentially proteolyzes eIF4GI and eIF4GII in a time- and dose-dependent manner. This system is based on the electroporation of an in vitro transcribed mRNA that contains the encephalomyocarditis virus internal ribosome entry site followed by the sequence of poliovirus 2Apro. In contrast to HeLa cells, expression of this protease in BHK-21 cells induces delayed hydrolysis kinetics of eIF4GI with respect to eIF4GII. Moreover, under these conditions the polyadenylate binding protein is not cleaved. Interestingly, translation of de novo synthesized luciferase mRNA is highly dependent on eIF4GI integrity, whereas ongoing translation is inhibited at the same time as eIF4GII cleavage. Moreover, reinitiation of a preexisting mRNA translation after polysome run-off is dependent on the integrity of eIF4GII. Notably, de novo translation of heat shock protein 70 mRNA depends little on eIF4GI integrity but is more susceptible to eIF4GII hydrolysis. Finally, translation of an mRNA containing encephalomyocarditis virus internal ribosome entry site when the two isoforms of eIF4G are differentially hydrolyzed has been examined.

Highlights

The initiation of translation is a major target for the regulation of gene expression in eukaryotic cells
Transfection of HeLa Cells with mRNAs Containing the PV 2A Sequence—We initially constructed a plasmid that encoded for the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) followed by the PV 2Apro sequence [21]
Electroporation of 9 ␮g of this mRNA was sufficient to drastically inhibit translation in HeLa cells accompanied by hydrolysis of eIF4G (Fig. 1, A and B)

Summary

Introduction

The initiation of translation is a major target for the regulation of gene expression in eukaryotic cells. Proteolysis kinetics of eIF4GII were delayed with respect to cleavage of eIF4GI in poliovirus (PV)and human rhinovirus-infected cells. In this regard disappearance of intact eIF4GII correlated with the abrogation of host translation in PV- and rhinovirus-infected and apoptotic cells (14 –16). Cleavage of both eIF4GI and eIF4GII strongly blocked the initiation of de novo synthesized mRNAs [17]. Differential Cleavage of eIF4GI and eIF4GII hydrolysis of PABP by 3Cpro in the absence of eIF4G degradation blocks the translation of endogenous mRNAs in HeLa cell extracts [18]. The effect of differential degradation of each eIF4G isoform in the translation of cellular mRNAs in culture cells has been examined

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Conclusion