Cap-Poly(A) Synergy in Mammalian Cell-free Extracts

Michel, Yanne; Poncet, Didier; Piron, Maria; Kean, Katherine M.; Borman, Andrew M.

doi:10.1074/jbc.m004304200

Cited by 140 publications

(60 citation statements)

References 33 publications

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“…Recent data have shown that another mechanism related to in vivo translational initiation, the interaction between the cap and poly(A) tail of eukaryotic mRNAs, does not occur in Flexi rabbit reticulocyte lysates. Instead, the mechanism operates in a modified system based on ribosome-depleted rabbit reticulocyte lysates (29,30). Alternatively, the translation of the ARFP/F/coreϩ1 protein may be regulated by different mechanisms depending on the cellular conditions and the ways in which the translation machinery is modified during HCV infection.…”

Section: Discussionmentioning

confidence: 99%

Two Alternative Translation Mechanisms Are Responsible for the Expression of the HCV ARFP/F/Core+1 Coding Open Reading Frame

Vassilaki

Mavromara

2003

Journal of Biological Chemistry

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HCV-1 produces a novel protein, known as ARFP, F, or core؉1. This protein is encoded by an open reading frame (ORF) that overlaps the core gene in the ؉1 frame (core؉1 ORF). In vitro this protein is produced by a ribosomal frameshift mechanism. However, similar studies failed to detect the ARFP/F/core؉1 protein in the HCV-1a (H) isolate. To clarify this issue and to elucidate the functions of this protein, we examined the expression of the core؉1 ORF by the HCV-1 and HCV-1a (H) isolates in vivo, in transfected cells. For this purpose, we carried out luciferase (LUC) tagging experiments combined with site-directed mutagenesis studies. Our results showed that the core؉1-LUC chimeric protein was efficiently produced in vivo by both isolates. More importantly, neither changes in the specific 10-A residue region of HCV-1 (codons 8 -11), the proposed frameshift site for the production of the ARFP/F/core؉1 protein in vitro, nor the alteration of the ATG start site of the HCV polyprotein to a stop codon significantly affected the in vivo expression of the core؉1 ORF. Furthermore, we showed that efficient translation initiation of the core؉1 ORF is mediated by internal initiation codon(s) within the core/core؉1-coding sequence, located between nucleotides 583 and 606. Collectively, our data suggest the existence of an alternative translation initiation mechanism that may result in the synthesis of a shorter form of the core؉1 protein in transfected cells.

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Section: Discussionmentioning

confidence: 99%

Two Alternative Translation Mechanisms Are Responsible for the Expression of the HCV ARFP/F/Core+1 Coding Open Reading Frame

Vassilaki

Mavromara

2003

Journal of Biological Chemistry

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“…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.…”

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confidence: 99%

“…The recent demonstrations that plant, yeast, and mammalian PABP can interact physically with the N-terminal part of eIF4G (11)(12)(13) and that capped polyadenylated mRNAs can be physically circularized in vitro by the addition of purified eIF4E, eIF4G and PABP (14) suggest that cap-poly(A) cooperativity somehow results from the physical interactions between the mRNA 5Ј and 3Ј ends (9,15). Indeed, disruption of the eIF4G-PABP interaction abolishes both the positive effects of poly(A) on uncapped mRNA translation in yeast (16) and abrogates cap-poly(A) synergy in cellfree mammalian extracts (17).…”

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confidence: 99%

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

Borman

Michel²,

Malnou³

et al. 2002

Journal of Biological Chemistry

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The 5 cap and 3 poly(A) tail of classical eukaryotic mRNAs functionally communicate to synergistically enhance translation initiation. Synergy has been proposed to result in part from facilitated ribosome recapture on circularized mRNAs. Here, we demonstrate that this is not the case. In poly(A)-dependent, ribosome-depleted rabbit reticulocyte lysates, the addition of exogenous poly(A) chains of physiological length dramatically stimulated translation of a capped, nonpolyadenylated mRNA. When the poly(A):RNA ratio approached 1, exogenous poly(A) stimulated translation to the same extent as the presence of a poly(A) tail at the mRNA 3 end. In addition, exogenous poly(A) significantly improved translation of capped mRNAs carrying short poly(A 50 ) tails. Trans stimulation of translation by poly(A) required the eIF4G-poly(A)-binding protein interaction and resulted in increased affinity of eIF4E for the mRNA cap, exactly as we recently described for cap-poly(A) synergy. These results formally demonstrate that mRNA circularization per se is not the cause of cap-poly(A) synergy at least in vitro.The 5Ј and 3Ј ends of the vast majority of eukaryotic mRNAs are modified post-transcriptionally by the addition, respectively, of a methylated m 7 GpppN cap structure and a poly(A) tail (1) Under appropriate competitive translation conditions, the 5Ј cap and 3Ј poly(A) tail synergistically promote translation initiation (6 -10), in a process requiring the poly(A)-binding protein (PABP) (11). The recent demonstrations that plant, yeast, and mammalian PABP can interact physically with the N-terminal part of eIF4G (11-13) and that capped polyadenylated mRNAs can be physically circularized in vitro by the addition of purified eIF4E, eIF4G and PABP (14) suggest that cap-poly(A) cooperativity somehow results from the physical interactions between the mRNA 5Ј and 3Ј ends (9, 15). Indeed, disruption of the eIF4G-PABP interaction abolishes both the positive effects of poly(A) on uncapped mRNA translation in yeast (16) and abrogates cap-poly(A) synergy in cellfree mammalian extracts (17).In mechanistic terms, the interaction of wheat germ PABP with eIF4F has been shown to increase the affinity of the eIF4F complex for cap analogue by some 40-fold (18). Similarly, the eIF4G-PABP interaction increases the functional affinity of eIF4E for the capped 5Ј end of polyadenylated mRNAs in mammalian extracts (19). Furthermore, the affinity of eIF4F-complexed plant PABP for poly(A) is significantly greater than that of free PABP (12). Thus, it seems probable that mRNA 5Ј-3Ј end cross-talk enhances translation, at least in part, by stimulating the formation of initiation factor-mRNA complexes. Because this process only requires a series of RNA-protein and protein-protein interactions, it could occur, theoretically, in trans.Several further potential translational advantages could be envisaged to result from mRNA circularization. For instance, the PABP-eIF4G interaction would provide a means of tethering the eIF4F complex to a mRNA (albeit at the ...

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“…Such a mechanism was demonstrated in yeast, plant, and mammalian systems (2,3) and plays a key role during development of Xenopus, Drosophila, and mouse (4). Translational synergy was also recapitulated in vitro (5)(6)(7)(8)(9). Hence, the cap-poly(A) tail synergy represents a common paradigm for translational control.…”

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confidence: 83%

A mechanism of translational repression by competition of Paip2 with eIF4G for poly(A) binding protein (PABP) binding

Karim

Svitkin

Kahvejian

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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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.circularization ͉ translation initiation T ranslational control is an important means by which cells govern gene expression. Initiation, the rate-limiting step of translation, is often the target of translational control (1). This control involves in many circumstances the cap structure at the mRNA 5Ј end and the poly(A) tail at the mRNA 3Ј end. Although both mRNA terminal structures stimulate translation on their own, their combined translational enhancement is synergistic. Such a mechanism was demonstrated in yeast, plant, and mammalian systems (2, 3) and plays a key role during development of Xenopus, Drosophila, and mouse (4). Translational synergy was also recapitulated in vitro (5-9). Hence, the cap-poly(A) tail synergy represents a common paradigm for translational control.The mechanism by which the mRNA 3Ј poly(A) tail synergizes with the 5Ј cap to stimulate translation was first documented in yeast, where PABP was shown to interact directly with the eIF4G subunit of the cap-binding complex eIF4F (see below) (10, 11). In yeast, PABP is an essential protein because deletion of the PABP1 gene is lethal (12). However, because deletion of the eIF4G binding site in PABP causes only a mild effect on yeast cell growth (13), it is not clear that PABP-eIF4G interaction is the only mechanism by which PABP regulates translation and cell growth in yeast. In addition, Searfoss et al. (14) demonstrated that yeast Ski proteins inhibit the activity of eIF5 and eIF4B in 60S ribosomal subunit joining and that this inhibition is reversed by PABP. These results implicate an important role for PABP in 60S ribosomal subunit joining. In contrast to yeast, the expression of an eIF4G mutant that does not interact with PABP in Xenopus oocytes repressed translation of polyadenylated mRNAs and inhibited progesteroneinduced oocyte maturation (15).PABP contains four RNA-recognition motifs (RRMs) and a proline-rich C-terminal region, which is N-terminal to a highly evolutionarily conserved sequence termed PABC (see below) (16,17). eIF4F is composed of the cap-binding subunit eIF4E, an RNA-dependent ATPase͞ATP-dependent RNA helicase, eIF4A, and eIF4G (reviewed in ref. 18). The latter serves...

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Cap-Poly(A) Synergy in Mammalian Cell-free Extracts

Cited by 140 publications

References 33 publications

Two Alternative Translation Mechanisms Are Responsible for the Expression of the HCV ARFP/F/Core+1 Coding Open Reading Frame

Two Alternative Translation Mechanisms Are Responsible for the Expression of the HCV ARFP/F/Core+1 Coding Open Reading Frame

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

A mechanism of translational repression by competition of Paip2 with eIF4G for poly(A) binding protein (PABP) binding

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