Differential cleavage of eIF4GI and eIF4GII in mammalian cells. Effects on translation.
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.
Castelló
Alfredo
A
Centro de Biología Molecular Severo Ochoa, Facultad de Ciencias and Centro Nacional de Biotecnología Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Alvarez
Enrique
E
Carrasco
Luis
L
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D039603:Eukaryotic Initiation Factor-4G / Q000235:genetics* / Q000378:metabolism*; D017930:Genes, Reporter / Q000235:genetics; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D014176:Protein Biosynthesis / Q000235:genetics*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism
D039603:Eukaryotic Initiation Factor-4G; D006360:Heat-Shock Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger