Nuclear Localization of Cytoplasmic Poly(A)-Binding Protein upon Rotavirus Infection Involves the Interaction of NSP3 with eIF4G and RoXaN
Maya HarbMichelle M. BeckerDamien VitourCarolina Hilma BaronPatrice VendeSpencer BrownSusanne BolteStefan T. AroldDidier Poncet
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Abstract:
Rotavirus nonstructural protein NSP3 interacts specifically with the 3' end of viral mRNAs, with the eukaryotic translation initiation factor eIF4G, and with RoXaN, a cellular protein of yet-unknown function. By evicting cytoplasmic poly(A) binding protein (PABP-C1) from translation initiation complexes, NSP3 shuts off the translation of cellular polyadenylated mRNAs. We show here that PABP-C1 evicted from eIF4G by NSP3 accumulates in the nucleus of rotavirus-infected cells. Through modeling of the NSP3-RoXaN complex, we have identified mutations in NSP3 predicted to interrupt its interaction with RoXaN without disturbing the NSP3 interaction with eIF4G. Using these NSP3 mutants and a deletion mutant unable to associate with eIF4G, we show that the nuclear localization of PABP-C1 not only is dependent on the capacity of NSP3 to interact with eIF4G but also requires the interaction of NSP3 with a specific region in RoXaN, the leucine- and aspartic acid-rich (LD) domain. Furthermore, we show that the RoXaN LD domain functions as a nuclear export signal and that RoXaN tethers PABP-C1 with RNA. This work identifies RoXaN as a cellular partner of NSP3 involved in the nucleocytoplasmic localization of PABP-C1.Keywords:
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A recent study revealed that poly(A)-binding protein (PABP) bound to poly(A) RNA exhibits a sharply bent configuration at the linker region between RNA-recognition motif 2 (RRM2) and RRM3, whereas free PABP exhibits a highly flexible linear configuration. However, the physiological role of the bent structure of mRNA-bound PABP remains unknown. We investigated a role of the bent structure of PABP by constructing a PABP variant that fails to form the poly(A)-dependent bent structure but maintains its poly(A)-binding activity. We found that the bent structure of PABP/poly(A) complex is required for PABP's efficient interaction with eIF4G and eIF4G/eIF4E complex. Moreover, the mutant PABP had compromised translation activation function and failed to augment the formation of 80S translation initiation complex in an in vitro translation system. These results suggest that the bent conformation of PABP, which is induced by the interaction with 3′ poly(A) tail, mediates poly(A)-dependent translation by facilitating the interaction with eIF4G and the eIF4G/eIF4E complex. The preferential binding of the eIF4G/eIF4E complex to the bent PABP/poly(A) complex seems to be a mechanism discriminating the mRNA-bound PABPs participating in translation from the idling mRNA-unbound PABPs.
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The eukaryotic mRNA 3' poly(A) tail and its associated poly(A)-binding protein (Pab1p) are important regulators of gene expression. One role for this complex in the yeast Saccharomyces cerevisiae is in translation initiation through an interaction with a 115-amino-acid region of the translation initiation factor eIF4G. The eIF4G-interacting domain of Pab1p was mapped to its second RNA recognition motif (RRM2) in an in vitro binding assay. Moreover, RRM2 of Pab1p was required for poly(A) tail-dependent translation in yeast extracts. An analysis of a site-directed Pab1p mutation which bound to eIF4G but did not stimulate translation of uncapped, polyadenylated mRNA suggested additional Pab1p-dependent events during translation initiation. These results support the model that the association of RRM2 of yeast Pab1p with eIF4G is a prerequisite for the poly(A) tail to stimulate the translation of mRNA in vitro.
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The bulk of cellular proteins derive from the translation of eukaryotic translation initiation factor (eIF)4E-bound mRNA. However, recent studies of nonsense-mediated mRNA decay (NMD) indicate that cap-binding protein (CBP)80-bound mRNA, which is a precursor to eIF4E-bound mRNA, can also be translated during a pioneer round of translation. Here, we report that the pioneer round, which can be assessed by measuring NMD, is not inhibited by 4E-BP1, which is known to inhibit steady-state translation by competing with eIF4G for binding to eIF4E. Therefore, at least in this way, the pioneer round of translation is distinct from steady-state translation. eIF4GI, poly(A)-binding protein (PABP)1, eIF3, eIF4AI, and eIF2α coimmunopurify with both CBP80 and eIF4E, which suggests that each factor functions in both modes of translation. Consistent with roles for PABP1 and eIF2α in the pioneer round of translation, PABP-interacting protein 2, which is known to destabilize PABP1 binding to poly(A) and inhibit steady-state translation, as well as inactive eIF2α, which is also known to inhibit steady-state translation, also inhibit NMD. Polysome profiles indicate that CBP80-bound mRNAs are translated less efficiently than their eIF4E-bound counterparts.
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The eukaryotic mRNA 3' poly(A) tail and the 5' cap cooperate to synergistically enhance translation. This interaction is mediated by the cap-binding protein eIF4E, the poly(A) binding protein (PABP), and eIF4G, a scaffolding protein that bridges between eIF4E and PABP to bring about the circularization of the mRNA. The translational repressor, Paip2 (PABP-interacting protein 2), inhibits translation by promoting the dissociation of PABP from poly(A). Here we report on the existence of an alternative mechanism by which Paip2 inhibits translation by competing with eIF4G for binding to PABP. We demonstrate that Paip2 can abrogate the translational activity of PABP, which is tethered to the 3' end of the mRNA. Thus, Paip2 can inhibit translation by a previously unrecognized mechanism, which is independent of its ability to disrupt PABP-poly(A) interaction.
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ABSTRACT Through its interaction with the 5′ translation initiation factor eIF4G, poly(A) binding protein (PABP) facilitates the translation of 5′-capped and 3′-poly(A)-tailed mRNAs. Rotavirus mRNAs are capped but not polyadenylated, instead terminating in a 3′ GACC motif that is recognized by the viral protein NSP3, which competes with PABP for eIF4G binding. Upon rotavirus infection, viral, GACC-tailed mRNAs are efficiently translated, while host poly(A)-tailed mRNA translation is, in contrast, severely impaired. To explore the roles of NSP3 in these two opposing events, the translational capabilities of three capped mRNAs, distinguished by either a GACC, a poly(A), or a non-GACC and nonpoly(A) 3′ end, have been monitored after electroporation of cells expressing all rotavirus proteins (infected cells) or only NSP3 (stably or transiently transfected cells). In infected cells, we found that the magnitudes of translation induction (GACC-tailed mRNA) and translation reduction [poly(A)-tailed mRNA] both depended on the rotavirus strain used but that translation reduction not genetically linked to NSP3. In transfected cells, even a small amount of NSP3 was sufficient to dramatically enhance GACC-tailed mRNA translation and, surprisingly, to slightly favor the translation of both poly(A)- and nonpoly(A)-tailed mRNAs, likely by stabilizing the eIF4E-eIF4G interaction. These data suggest that NSP3 is a translational surrogate of the PABP-poly(A) complex; therefore, it cannot by itself be responsible for inhibiting the translation of host poly(A)-tailed mRNAs upon rotavirus infection. IMPORTANCE To control host cell physiology and to circumvent innate immunity, many viruses have evolved powerful mechanisms aimed at inhibiting host mRNA translation while stimulating translation of their own mRNAs. How rotavirus tackles this challenge is still a matter of debate. Using rotavirus-infected cells, we show that the magnitude of cellular poly(A) mRNA translation differs with respect to rotavirus strains but is not genetically linked to NSP3. Using cells expressing rotavirus NSP3, we show that NSP3 alone not only dramatically enhances rotavirus-like mRNA translation but also enhances poly(A) mRNA translation rather than inhibiting it, likely by stabilizing the eIF4E-eIF4G complex. Thus, the inhibition of cellular polyadenylated mRNA translation during rotavirus infection cannot be attributed solely to NSP3 and is more likely the result of global competition between viral and host mRNAs for the cellular translation machinery.
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Protein synthesis in eukaryotes initiates with binding of the multisubunit translation initiation complex eIF4F. This complex contains eIF4E, eIF4A and eIF4G. eIF4E directly interacts with the cap structure, eIF4A is an RNA helicase and eIF4G acts as a scaffold for the complex. eIF4G contains the binding sites for both the subunits i.e., eIF4A and eIF4E and it also interacts with poly(A)-binding protein (PABP). In present study we have identified and characterized the main components of the eIF4F complex i.e., eIF4E, eIF4A and eIF4G and PABP from Plasmodium falciparum. Molecular modeling of PfeIF4E, PfeIF4G and PfPABP confirms that they contain all the characteristic conserved structural features. We have annotated some of the genes of P. falciparum and as a result these studies demonstrate that the components of translation initiation complex are highly conserved. Therefore these studies will contribute to understand the basic biology and components of translation complex in P. falciparum.
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Protein synthesis is a major determinate in cell growth and differentiation. Here, we investigated the porcine uterine tissue during early pregnancy. The objective was the analysis of the initiation of translation, which is believed to be the rate-limiting process of protein synthesis. Performing Western blot screening during implantation and placentation, in vitro protease assays and mass spectrometry, we revealed that essential components of the cap-binding complex eIF4F and its regulators are targets of specific proteolytically processing. This proteolytical activity shifts from the implantation site to the peri-placental area later in development. The results show that the cap-binding protein eIF4E is N-terminally truncated which results in lower binding to the repressors 4E-BP1 and 4E-BP2. On the other hand, 4E-BP1 is mainly degraded, whereas 4E-BP2 emerges as N-terminally truncated stable fragments. It is likely that these fragments have lost an essential sequence motif that enables the phosphorylation and inactivation of the repressor. Furthermore, the Akt/mTor signaling cascade (which phosphorylates 4E-BP1 and 2) but not the MAPK pathway (which phosphorylated eIF4E and together with Akt/mTor hierarchically 4E-BP1 and 2) was inactivated. This results in long-term translation repression of eIF4E-sensitive and 4E-BP2-dependent mRNAs. However, other translation initiation factors like eIF4A, eIF2α, and the poly(A)-binding protein PABP are not modified. In conclusion, we suggest that the observed specific modification of the mRNA-cap-binding complex in the porcine endometrium during early pregnancy is essential to regulate gene expression to establish the noninvasive implantation and to sustain the ongoing gravidity.
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