Biochemical characterisation of cap-poly(A) synergy in rabbit reticulocyte lysates: the eIF4G-PABP interaction increases the functional affinity of eIF4E for the capped mRNA 5'-end

Borman, Andrew M.; Michel, Yanne; Kean, Katherine M.

doi:10.1093/nar/28.21.4068

Cited by 103 publications

(95 citation statements)

References 33 publications

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“…The recently described nuclease-treated, ribosomedepleted RRL translation system is ideal for such a study, because poly(A)-dependence results from physically limiting concentrations of ribosomes and ribosome-associated translation factors rather than the presence of intact, functional competitor mRNAs (19). Effectively, functional analyses of the mechanism of poly(A) action are hindered in competitor-based systems by the fact that poly(A)-dependence of the extract is lost as soon as translation conditions are altered (7,17,19). The pOp24 plasmids used for the present study have been described elsewhere (17).…”

Section: Resultsmentioning

confidence: 99%

“…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).…”

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confidence: 99%

“…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%

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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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Section: Resultsmentioning

confidence: 99%

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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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“…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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“…3). This interaction between the N terminus of eIF4G and PABP has been reported to induce an increase of the affinity of the cap-binding protein eIF4E for the cap structure (76) (see also below). In some in vitro systems, investigators have also seen stimulated translation of capped mRNAs when poly(A) was added in trans, raising the possibility that there may not be a mandatory requirement for the poly(A)-PABP-eIF4G-eIF4E-cap interaction to occur in cis, (66,77) although this phenomenon seems difficult to place into the physiological context of cellular mRNA translation.…”

Section: Assembly Of the 80s Ribosomementioning

confidence: 91%

Starting the protein synthesis machine: eukaryotic translation initiation

2003

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SummaryThe final assembly of the protein synthesis machinery occurs during translation initiation. This delicate process involves both ends of eukaryotic messenger RNAs as well as multiple sequential protein-RNA and protein-protein interactions. As is expected from its critical position in the gene expression pathway between the transcriptome and the proteome, translation initiation is a selective and highly regulated process. This synopsis summarises the current status of the field and identifies intriguing open questions.

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Biochemical characterisation of cap-poly(A) synergy in rabbit reticulocyte lysates: the eIF4G-PABP interaction increases the functional affinity of eIF4E for the capped mRNA 5'-end

Cited by 103 publications

References 33 publications

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

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

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

Starting the protein synthesis machine: eukaryotic translation initiation

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