Interaction of a limited set of proteins with different mRNAs and protection of 5′-caps against pyrophosphatase digestion in initiation complexes

Sonenberg, Nahum; Morgan, M.; Testa, Douglas; Colonno, Richard J.; Shatkin, Aaron J.

doi:10.1093/nar/7.1.15

Cited by 45 publications

(24 citation statements)

References 38 publications

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“…Cross-linking of the cap (Sonenberg et al 1979a) and the nucleotide at the +2 position from the cap (Lindqvist et al 2008) with an eIF3 subunit of a similar molecular weight has been reported previously and was attributed to eIF3d (Lindqvist et al 2008). We, however, identified the cap-interacting subunit as eIF3l.…”

Section: Translation Initiation On Capped Mrnassupporting

confidence: 75%

“…Consistently, cross-linking of the cap and eIF4E is inefficient in cellfree extracts (Sonenberg and Shatkin 1977;Sonenberg et al 1979a), and it was also suggested that, during initiation, the eIF4E-cap interaction is disrupted (Sonenberg et al 1979b). According to our model, the most likely stage at which eIF4E is released from the cap is the beginning of attachment, when dissociation would be required to avoid steric hindrance as mRNA enters the mRNAbinding channel.…”

Section: Translation Initiation On Capped Mrnassupporting

confidence: 67%

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Toward the mechanism of eIF4F-mediated ribosomal attachment to mammalian capped mRNAs

2016

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Ribosomal attachment to mammalian capped mRNAs is achieved through the cap-eukaryotic initiation factor 4E (eIF4E)-eIF4G-eIF3-40S chain of interactions, but the mechanism by which mRNA enters the mRNA-binding channel of the 40S subunit remains unknown. To investigate this process, we recapitulated initiation on capped mRNAs in vitro using a reconstituted translation system. Formation of initiation complexes at 5 ′ -terminal AUGs was stimulated by the eIF4E-cap interaction and followed "the first AUG" rule, indicating that it did not occur by backward scanning. Initiation complexes formed even at the very 5 ′ end of mRNA, implying that Met-tRNA i Met inspects mRNA from the first nucleotide and that initiation does not have a "blind spot." In assembled initiation complexes, the cap was no longer associated with eIF4E. Omission of eIF4A or disruption of eIF4E-eIF4G-eIF3 interactions converted eIF4E into a specific inhibitor of initiation on capped mRNAs. Taken together, these results are consistent with the model in which eIF4E-eIF4G-eIF3-40S interactions place eIF4E at the leading edge of the 40S subunit, and mRNA is threaded into the mRNA-binding channel such that Met-tRNA i Met can inspect it from the first nucleotide. Before entering, eIF4E likely dissociates from the cap to overcome steric hindrance. We also found that the m 7 G cap specifically interacts with eIF3l.[Keywords: eukaryotic translation initiation; eIF4E; eIF4F; m 7 G; eIF3l; 40S ribosomal subunit]Supplemental material is available for this article.Received April 7, 2016; revised version accepted June 1, 2016.Translation initiation on the majority of mammalian cellular mRNAs occurs by the scanning mechanism (Jackson et al. 2010). First, eukaryotic initiation factor 2 (eIF2), GTP, and Met-tRNA i Met form a ternary complex (eIF2-TC), which, with the multisubunit eIF3 and monomeric eIF1 and eIF1A, binds to the 40S ribosomal subunit, yielding a 43S preinitiation complex. Attachment of the 43S complex to capped mRNA is mediated by eIF4A, eIF4B, and eIF4F. eIF4F comprises the cap-binding protein eIF4E, the DEAD-box RNA helicase eIF4A (which also exists in a free form), and the eIF4G scaffold, which interacts with the other two subunits as well as eIF3. eIF4A's helicase activity is stimulated by both eIF4G and eIF4B. Group 4 eIFs cooperatively unwind the cap-proximal region of mRNA, preparing it for attachment of 43S complexes, which is promoted by the eIF4G-eIF3 interaction. After attachment, 43S complexes scan to the first AUG codon in a favorable nucleotide context, where they form 48S initiation complexes with established codonanti-codon base-pairing. Group 4 eIFs also assist 43S complexes during scanning. Scanning on mRNAs with highly structured 5 ′ untranslated regions (UTRs) additionally requires DHX29, a DExH-box protein that interacts directly with 40S subunits. eIF1, in cooperation with eIF1A, ensures the fidelity of initiation codon selection, discriminating against initiation at non-AUG codons and AUGs that are too close to the 5 ′ end of mRN...

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Section: Translation Initiation On Capped Mrnassupporting

confidence: 75%

Section: Translation Initiation On Capped Mrnassupporting

confidence: 67%

Toward the mechanism of eIF4F-mediated ribosomal attachment to mammalian capped mRNAs

2016

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“…eIF3 can be UV cross-linked to ␤-globin and other mRNAs in binary complexes (Fig. 3F, lane 1; Sonenberg et al 1979;Setyono et al 1984;Westermann and Nygard 1984). It can also be UV cross-linked to […”

Section: Stimulation Of Stable Binding Of Eif3j− To 40s Subunits By Omentioning

confidence: 99%

Binding of eukaryotic initiation factor 3 to ribosomal 40S subunits and its role in ribosomal dissociation and anti-association

Kolupaeva¹,

Unbehaun²,

Lomakin³

et al. 2005

RNA

115

134

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The multisubunit eukaryotic initiation factor (eIF) 3 plays various roles in translation initiation that all involve interaction with 40S ribosomal subunits. eIF3 can be purified in two forms: with or without the loosely associated eIF3j subunit (eIF3j+ and eIF3j−, respectively). Although unlike eIF3j+, eIF3j− does not bind 40S subunits stably enough to withstand sucrose density gradient centrifugation, we found that in addition to the known stabilization of the eIF3/40S subunit interaction by the eIF2·GTP·Met-tRNA i Met ternary complex, eIF3j−/40S subunit complexes were also stabilized by single-stranded RNA or DNA cofactors that were at least 25 nt long and could be flanked by stable hairpins. Of all homopolymers, oligo(rU), oligo(dT), and oligo(dC) stimulated the eIF3/40S subunit interaction, whereas oligo(rA), oligo(rG), oligo(rC), oligo(dA), and oligo(dG) did not. Oligo(U) or oligo(dT) sequences interspersed by other bases also promoted this interaction. The ability of oligonucleotides to stimulate eIF3/40S subunit association correlated with their ability to bind to the 40S subunit, most likely to its mRNA-binding cleft. Although eIF3j+ could bind directly to 40S subunits, neither eIF3j− nor eIF3j+ alone was able to dissociate 80S ribosomes or protect 40S and 60S subunits from reassociation. Significantly, the dissociation/anti-association activities of both forms of eIF3 became apparent in the presence of either eIF2-ternary complexes or any oligonucleotide cofactor that promoted eIF3/40S subunit interaction. Ribosomal dissociation and anti-association activities of eIF3 were strongly enhanced by eIF1. The potential biological role of stimulation of eIF3/40S subunit interaction by an RNA cofactor in the absence of eIF2-ternary complex is discussed.

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“…Exceptions are the RNAs of picornaviruses (2-4) and some plant viruses (5,6). In crude protein-synthesizing systems several polypeptides (cap-binding proteins, CBPs) were shown to crosslink specifically to the cap structure (7). In addition, a Mr 24,000 polypeptide that binds to the cap structure has been identified in partially purified initiation factor fractions (8) and purified by affinity chromatography on m7GDP-Sepharose (9).…”

mentioning

confidence: 99%

Association of a Mr 50,000 cap-binding protein with the cytoskeleton in baby hamster kidney cells.

Zumbe¹,

Stähli²,

Trachsel³

1982

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

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A monoclonal antibody directed against eukaryotic mRNA 5'-cap-binding protein (anti-CBP antibody) was used to localize cap-binding protein (CBP) in BHK-21 baby hamster kidney cells by immunofluorescence microscopy. It was found that the antibody reacts with a fibrous network extending through the cytoplasm in a radial arrangement. The network behaves like intermediate filaments in colchicine-treated cells, suggesting a direct or indirect linkage of CBP with intermediate filaments. The association of CBP with a cytoskeletal element was further confirmed by isolation of proteins from Triton X-100-extracted cells and identification of CBP in the cytoskeletal fraction with anti-CBP antibody. The major polypeptide reacting with anti-CBP antibody is a Mr 50,000 component. Tryptic peptide mapping showed that this polypeptide is related to a Mr 24,000 polypeptide identified as CBP in earlier experiments [Sonenberg, N., Morgan, M.

show abstract

Interaction of a limited set of proteins with different mRNAs and protection of 5′-caps against pyrophosphatase digestion in initiation complexes

Cited by 45 publications

References 38 publications

Toward the mechanism of eIF4F-mediated ribosomal attachment to mammalian capped mRNAs

Toward the mechanism of eIF4F-mediated ribosomal attachment to mammalian capped mRNAs

Binding of eukaryotic initiation factor 3 to ribosomal 40S subunits and its role in ribosomal dissociation and anti-association

Association of a Mr 50,000 cap-binding protein with the cytoskeleton in baby hamster kidney cells.

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