Differential Cleavage of eIF4GI and eIF4GII in Mammalian Cells

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 timeand 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.

RNA nuclear export is blocked by poliovirus 2A protease and is concomitant with nucleoporin cleavage

“…5A). Consistent with previous observations (Castello et al, 2006a), inhibition of cellular translation correlated with the proteolysis of eIF4GI and eIF4GII (Fig. 5A,B).…”

“…pro alters nuclear export of mRNAs, U snRNAs and rRNAs but not tRNAs Translation of newly synthesized mRNAs is blocked by PV 2A pro earlier than that of mRNAs involved in ongoing protein synthesis (Castello et al, 2006a;Novoa and Carrasco, 1999), suggesting that this protease might interfere with an early step of gene expression. Previous reports showed that the NPC was altered in PV-infected cells (Belov et al, 2004;Gustin and Sarnow, 2001; Gustin and Cytopathic viruses have developed successful strategies to block or, at least, to attenuate host interference with their replication.…”

“…First, we analyzed the cellular distribution of de-novosynthesized luciferase mRNAs in Tet-off HeLa X1/5 cells that express PV 2A pro (Castello et al, 2006a). Luciferase mRNA synthesis was induced by removal of tetracycline (Tet) from the culture medium , and the expression of PV 2A pro was achieved by the electroporation of 1 or 9 g of an in-vitro-synthesized mRNA that contained the encephalomyocarditis virus (EMCV) IRES followed by the PV 2A pro coding sequence (IRES-2A mRNA) (Castello et al, 2006a). At 8 hours postelectroporation (hpe), luciferase activity and the integrity of eIF4GI and eIF4GII were estimated.…”

Mutation of Eif4g3, encoding a eukaryotic translation initiation factor, causes male infertility and meiotic arrest of mouse spermatocytes

“…A number of IRES-containing mRNAs are translated by a cap-independent mechanism. This might be the case for human HSPA mRNA, which is reported to contain an IRES element at its 5Јuntranslated region (Rubtosova et al, 2003) and is dependent on the integrity of EIF4G3 for in vitro translation (Castello et al, 2006). The mouse Hspa2 also has a long 5Ј untranslated leader sequence, although no common DNA sequence in the 5Ј untranslated region is shared between mouse Hspa2 and other Hspa genes (Dix et al, 1996b).…”

Translation initiation factor eIF4G1 preferentially binds yeast transcript leaders containing conserved oligo-uridine motifs

“…In the context of some viral mRNAs, direct RNA binding by eIF4G is both necessary and sufficient to confer efficient translation, even in the absence of a cap or a poly(A) tail (Pestova et al 1996a,b). High-affinity binding of eIF4G could facilitate the translation of those cellular mRNAs most dependent on the activity of the eIF4F complex, such as those with long or structured TLs (Sen et al 2015(Sen et al , 2016, or allow coordinated translational control under conditions that limit eIF4F activity including viral infection (Castelló et al 2006(Castelló et al , 2011, nutrient depletion (Thoreen et al 2012), and heat shock (Cuesta et al 2000).…”