Eukaryotic Initiation Factor 4G-Poly(A) Binding Protein Interaction Is Required for Poly(A) Tail-Mediated Stimulation of Picornavirus Internal Ribosome Entry Segment-Driven Translation but Not for X-Mediated Stimulation of Hepatitis C Virus Translation

Abstract

Efficient translation of most eukaryotic mRNAs results from synergistic cooperation between the 5 m 7 GpppN cap and the 3 poly(A) tail. In contrast to such mRNAs, the polyadenylated genomic RNAs of picornaviruses are not capped, and translation is initiated internally, driven by an extensive sequence termed IRES (for internal ribosome entry segment). Here we have used our recently described poly(A)-dependent rabbit reticulocyte lysate cell-free translation system to study the role of mRNA polyadenylation in IRESdriven translation. Polyadenylation significantly stimulated translation driven by representatives of each of the three types of picornaviral IRES (poliovirus, encephalomyocarditis virus, and hepatitis A virus, respectively). This did not result from a poly(A)-dependent alteration of mRNA stability in our in vitro translation system but was very sensitive to salt concentration. Disruption of the eukaryotic initiation factor 4G-poly(A) binding protein (eIF4G-PABP) interaction or cleavage of eIF4G abolished or severely reduced poly(A) tail-mediated stimulation of picornavirus IRES-driven translation. In contrast, translation driven by the flaviviral hepatitis C virus (HCV) IRES was not stimulated by polyadenylation but rather by the authentic viral RNA 3 end: the highly structured X region. X region-mediated stimulation of HCV IRES activity was not affected by disruption of the eIF4G-PABP interaction. These data demonstrate that the protein-protein interactions required for synergistic cooperativity on capped and polyadenylated cellular mRNAs mediate 3-end stimulation of picornaviral IRES activity but not HCV IRES activity. Their implications for the picornavirus infectious cycle and for the increasing number of identified cellular IRES-carrying mRNAs are discussed.The initiation of protein synthesis on most mRNAs in eukaryotes follows binding of the 40S ribosomal subunit near the capped 5Ј end of the mRNA and subsequent migration of this subunit along the mRNA in a 5Ј-to-3Ј direction until a suitable initiation codon is selected (for a review, see reference 29). Recognition of the mRNA 5Ј end and 40S subunit recruitment requires the eukaryotic initiation factor (eIF) 4F complex (for reviews, see references 35 and 43). The eIF4F complex comprises the cap binding protein (eIF4E) and an ATP-dependent RNA helicase (eIF4A) bound, respectively, toward the N and C termini of a scaffold protein, eIF4G (for a review, see references 14 and 35). The C-terminal half of eIF4G is also thought to associate with the multisubunit eIF3 complex, which binds the 40S ribosomal subunit directly thus bridging the gap between the mRNA 5Ј end and the 40S subunit (reviewed in reference 17).The vast majority of eukaryotic mRNAs are not only capped at their 5Ј end but are also polyadenylated at their 3Ј end. Aside from a role in mRNA metabolism (see reference 45 for a review), the poly(A) tail functions as a translational enhancer and interacts synergistically with the 5Ј cap to stimulate translation initiation (12,23,42,43). This co...

Differential Cleavage of eIF4GI and eIF4GII in Mammalian Cells

“…Cleavage of eIF4G by PV 2A pro separates its PABP binding domain, abolishing the stimulation provided by poly(A) tail (41,27). This behavior was also observed for polyadenylated picornavirus IRES-containing mRNAs (27,28). When eIF4G is hydrolyzed, the EMC-Luc and EMC-Luc-Poly(A) mRNAs exhibit a similar translatability since poly(A) tail does not contribute to mRNA translation.…”

“…pro (27,28). These data suggest that poly(A) tail does not contribute to translation initiation when eIF4G is proteolyzed.…”

“…The 5Ј-untranslated region of these mRNAs allows recruitment of the 40 S ribosomal subunit under conditions in which the eIF4F complex is disrupted by viral protein activity (2). Translation of picornavirus IRES-containing mRNAs is enhanced by the presence of a poly(A) tail at its 3Ј end (27,28). EMCV IRES has been classified as type II on the basis of its primary sequence and secondary structure conservation and its requirements for optimal internal initiation in vitro (29).…”

Free Poly(A) Stimulates Capped mRNA Translation in Vitro through the eIF4G-Poly(A)-binding Protein Interaction

“…Additionally, the observed effects were specific to poly(A), as evidenced by the inability of either poly(dA) or poly(U) to effect similar stimulation of translation on either ϩ/Ϫ or ϩ/ϩ mRNAs (Table II) with the mRNA 3Ј poly(A) tail and effectively sequestering this element. Finally, exogenous poly(A) had no discernible effect on mRNA stability in the depleted RRL system (data not shown), which is perhaps not surprising given that poly(A) tails acting in cis stimulate translation in this system without altering mRNA half-life (17,22).…”

“…Because a competitive environment is assured by rendering translation components physically limiting, rather than by adding excess competitor mRNAs, this system allows the molecular dissection of the functional interactions between mRNA 5Ј and 3Ј ends (17,19,22). Here we describe the use of this system for a detailed study of the effect of exogenous poly(A) on translation of mRNAs carrying various combinations of 5Ј cap and 3Ј poly(A) tail.…”