Capped mRNAs with Reduced Secondary Structure Can Function in Extracts from Poliovirus-Infected Cells

Sonenberg, Nahum; Guertin, Denise; Lee, Kevin A.W.

doi:10.1128/mcb.2.12.1633

Cited by 64 publications

(25 citation statements)

References 40 publications

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“…This result is consistent with the partial resistance that this 5Ј-UTR confers to inhibition of translation by transdominant mutant eIF4A (Svitkin et al 2001) and with earlier findings that translation of mRNAs with unstructured 5Ј-UTRs has a low requirement for the cap-binding complex eIF4F (Sonenberg et al 1982;Gehrke et al 1983).…”

Section: Ribosomal Attachment and Scanning On An Unstructured 5ј-utr supporting

confidence: 92%

The roles of individual eukaryotic translation initiation factors in ribosomal scanning and initiation codon selection

Pestova¹,

Kolupaeva²

2002

Genes Dev.

498

632

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To elucidate an outline of the mechanism of eukaryotic translation initiation, 48S complex formation was analyzed on defined mRNAs in reactions reconstituted in vitro from fully purified translation components. We found that a ribosomal 40S subunit, eukaryotic initiation factor (eIF) 3, and the eIF2 ternary complex form a 43S complex that can bind to the 5-end of an unstructured 5-untranslated region (5-UTR) and in the presence of eIF1 scan along it and locate the initiation codon without a requirement for adenosine triphosphate (ATP) or factors (eIF4A, eIF4B, eIF4F) associated with ATP hydrolysis. Scanning on unstructured 5-UTRs was enhanced by ATP, eIFs 4A and 4B, and the central domain of the eIF4G subunit of eIF4F. Their omission increased the dependence of scanning on eIFs 1 and 1A. Ribosomal movement on 5-UTRs containing even weak secondary structures required ATP and RNA helicases. eIF4F was essential for scanning, and eIFs 4A and 4B were insufficient to promote this process in the absence of eIF4F. We report that in addition to its function in scanning, eIF1 also plays a principal role in initiation codon selection. In the absence of eIF1, 43S complexes could no longer discriminate between cognate and noncognate initiation codons or sense the nucleotide context of initiation codons and were able to assemble 48S complexes on 5-proximal AUG triplets located only 1, 2, and 4 nt from the 5-end of mRNA.

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Section: Ribosomal Attachment and Scanning On An Unstructured 5ј-utr supporting

confidence: 92%

The roles of individual eukaryotic translation initiation factors in ribosomal scanning and initiation codon selection

Pestova¹,

Kolupaeva²

2002

Genes Dev.

498

632

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“…is clearly less dependent on added eIF-4E for translation than are CAT mRNA and total yeast mRNA. The fact that AMV-4 RNA is efficiently translated under conditions of decreased cap-binding activity agrees with the earlier finding that AMV-4 RNA translation is relatively insensitive to cap analog inhibition (24). It is therefore possible that AMV-4 RNA does not require eIF-4E for translation.…”

Section: Resultssupporting

confidence: 78%

“…It is therefore possible that AMV-4 RNA does not require eIF-4E for translation. This conclusion is supported by the finding that AMV-4 RNA is efficiently translated in extracts derived from poliovirus-infected HeLa cells (10,24) in which cap-binding protein activity is dramatically reduced (for reviews, see references 11 and 23). AMV-4 RNA has little secondary structure in its 5'-noncoding region (13), and it was postulated that this feature allows translation under conditions of reduced capbinding protein activity (10,13).…”

Section: Resultssupporting

confidence: 71%

Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system.

Altmann¹,

Sonenberg²,

Trachsel³

1989

Mol. Cell. Biol.

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The gene encoding translation initiation factor 4E (eIF-4E) from Saccharomyces cerevisiae was randomly mutagenized in vitro. The In eucaryotes, ribosomes initiate translation of mRNA by binding at or near the 5'-terminal cap structure. Bound ribosomes then scan the mRNA in the 5' to 3' direction to find the first AUG codon in optimal sequence context (for reviews, see references 11, 14, and 19). Presumably, this cap-dependent pathway represents the major translation initiation pathway in eucaryotes. More recently, however, alternative initiation pathways have been described. (i) Ribosomes can reinitiate translation after termination of translation of an open reading frame (reinitiation) (15,17,20), and (ii) ribosomes can initiate translation internally, e.g., initiate at a downstream open reading frame without translating the upstream open reading frame (internal initiation) (21). Whereas the initiation factor requirements for cap-dependent translation are known (for reviews, see references 11 and 19), the mechanisms and factor requirements for reinitiation and internal initiation have yet to be determined. Since initiation factor functions appear to be conserved between higher and lower eucaryotes, as indicated by amino acid sequence similarities between mammalian and yeast (Saccharomyces cerevisiae) initiation factors (1, 2, 7, 16) and substitution of a mouse initiation factor for the homologous yeast factor in vivo (M. Altmann, P. P. Muller, J. Pelletier, N. Sonenberg, and H. Trachsel, J. Biol. Chem., in press), we use S. cerevisiae as a model system to study factor requirements for translation of different mRNAs. Here, we describe the construction of an initiation factor 4E (eIF-4E)-dependent system and provide evidence for eIF-4E-independent mRNA translation. (ii) In vitro mutagenesis. pMDA-101 (about 50 ,ug) was treated with 1 M hydroxylamine (neutralized by adding NaOH to a final concentration of 0.5 M)-10 mM EDTA at 65°C for 3 h. The transformation efficiency (LiOAc method) (3) of the mutagenized plasmid pool was determined to be about 15% of that of nonmutagenized plasmid. Mutagenized plasmid was subjected to extensive phenol extraction (at least five times) and two ethanol precipitations. MATERIALS AND METHODSIsolation of temperature-sensitive yeast strains. The mutagenized plasmid pool was transformed into T93C (a eIF-4E::LEU2 ura3 trpl leu2[pGAL1-eIF-4E URA3]), a haploid S. cerevisiae strain having its chromosomal copy of the eIF-4E gene removed and replaced by the LEU2 gene (3). In these cells, the eIF-4E function is provided by the plasmid pGAL1-eIF-4E (Altmann et al., in press

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“…As compared with native reovirus mRNA, initiation complex formation on I-substituted reovirus mRNA in the wheat germ system is more resistant to inhibition by high salt concentrations, or by antibodies against eIF-4F components, and somewhat more resistant to inhibition by cap analogues [28,341. Unlike native reovirus rnRNA, I-substituted mRNA could form initiation complexes in extracts of poliovirus-infected HeLa cells (which lack functional eIF-4F activity), whilst in extracts from uninfected cells, high salt concentrations were more inhibitory to initiation on native mRNA than on I-substituted reovirus mRNA [35]. All these differences between I-substituted and native reovirus mRNA are almost identical to the differences between AMV RNA 4 and other capped mRNAs, not only with respect to initiation complex formation assays, but also in cell-free translation assays [28, 33, 35, 361. What is striking is that secondary structure is correlated not only with the requirement for eIF-4F and a 5'-cap structure for efficient initiation, but also with the ATP dependence of events associated with initiation, which raises the possibility that the ATP requirement is mainly or exclusively related to the action of eIF-4F and other factors at the 5' end, presumably by virtue of their helicase action.…”

Section: Discussionmentioning

confidence: 99%

The ATP requirement for initiation of eukaryotic translation varies according to the mRNA species

Jackson

1991

European Journal of Biochemistry

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The requirement for ATP for initiation of eukaryotic mRNA translation was tested using gel-filtered rabbit reticulocyte lysates incubated with labelled Met-tRNA?"' and exogenous RNA templates, and assaying the formation of labelled 80s initiation complexes in the presence of GTP, or labelled 40s initiation complexes in the presence of a non-hydrolysable analogue of GTP. Initiation complex formation on globin mRNA, or on capped viral RNAs such as papaya mosaic virus RNA and tobacco mosaic virus RNA, was strongly stimulated by ATP. In contrast, initiation complex formation on (uncapped) encephalomyocarditis virus RNA was uninfluenced by the presence or absence of ATP, which may be correlated with the recent evidence for scanning-independent internal initiation on this viral RNA. In addition, initiation complex formation on uncapped cowpea mosaic virus RNA and on poly(A,U,G) was only slightly stimulated by ATP, much less than in the case of the capped RNAs. These results suggest that most of the ATP hydrolysed during translation initiation is consumed in cap-dependent processes, probably in unwinding the mRNA, and relatively little in the actual migration or scanning of 40s subunits along the mRNA.One of the striking differences between translation initiation in eukaryotes and prokaryotes is that the assembly of the initiation complex in eukaryotic systems requires ATP, specifically for the binding of 40s ribosomal pre-initiation complexes to the mRNA [l, 21. Although this role of ATP was first revealed in experiments using purified initiation factors in highly fractionated systems [l ,2], which have the disadvantage of a relatively low rate of protein synthesis, it has been confirmed using crude gel-filtered wheat germ extract and rabbit reticulocyte lysate [3 -61, two systems which synthesise protein at rates approaching those pertaining in vivo.When this ATP requirement was first discovered it presented a puzzle, for the fact that initiation in prokaryotic systems is independent of ATP indicates that the binding of the small ribosomal subunit to mRNA is not a process which intrinsically requires the hydrolysis of energy-rich bonds. The subsequent realisation that the mechanism of initiation site selection in eukaryotic systems is quite different, involving a scanning of the mRNA from the 5' end by the small ribosomal subunit [7, 81, Enzyme. Creatine phosphokinase (EC 2.7.3.2) migration or scanning of the 40s ribosomal subunits on mRNA in the wheat germ system was dependent on ATP hydrolysis [3,4]. More recent work in several laboratories has also implicated ATP hydrolysis in the action of two initiation factors which operate at the mRNA binding step: eIF-4F and eIF-4A (reviewed in [9]). These initiation factors are thought to potentiate an ATP-dependent unwinding of the mRNA initiated at the 5' end by eIF-4F, and continued in a 5'-3' direction by eIF-4A, with eIF-4B playing a role as a cocatalyst in conjunction with both eIF-4F and eIF-4A [lo, 111. This raises the question of whether the observed ATP requir...

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Capped mRNAs with Reduced Secondary Structure Can Function in Extracts from Poliovirus-Infected Cells

Cited by 64 publications

References 40 publications

The roles of individual eukaryotic translation initiation factors in ribosomal scanning and initiation codon selection

The roles of individual eukaryotic translation initiation factors in ribosomal scanning and initiation codon selection

Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system.

The ATP requirement for initiation of eukaryotic translation varies according to the mRNA species

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