RNA-binding activity of translation initiation factor eIF4G1 from <i>Saccharomyces cerevisiae</i>

Berset, Catherine; Zurbriggen, Andreas; Djafarzadeh, Siamak; Altmann, Michael; Trachsel, Hans

doi:10.1261/rna.5380903

Cited by 64 publications

(89 citation statements)

References 37 publications

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“…Although previous work suggested that eIF4G1 binds RNA nonspecifically (Berset et al 2003), certain viral mRNAs rely on high-affinity binding to eIF4G for their translation (Pestova et al 1996a), raising the possibility that specific cellular mRNAs might also exploit the intrinsic RNA-binding preferences of eIF4G. Here, we have systematically determined the RNA-binding preferences of eIF4G1 in vitro and discovered a strong and specific affinity of eIF4G1 for oligo(U) sequences.…”

Section: Discussionmentioning

confidence: 90%

“…The RNA-binding activity of eIF4G1 is essential for yeast growth (Berset et al 2003), yet the role of this activity in translation initiation is not currently understood. Although previous work suggested that eIF4G1 binds RNA nonspecifically (Berset et al 2003), certain viral mRNAs rely on high-affinity binding to eIF4G for their translation (Pestova et al 1996a), raising the possibility that specific cellular mRNAs might also exploit the intrinsic RNA-binding preferences of eIF4G.…”

Section: Discussionmentioning

confidence: 99%

“…eIF4G bridges the cap-binding activity of eIF4E, the helicase activity of eIF4A, and the poly-A binding activity of PABP/Pab1, thereby forming the translational "closed loop" complex (for review, see Thompson and Gilbert 2017). eIF4G contains three RNA-binding domains that directly interact with mRNA and are essential for yeast growth (Berset et al 2003;Park et al 2011a), although specific functional interactions between eIF4G and cellular mRNAs have not been characterized. In the context of some viral mRNAs, direct RNA binding by eIF4G is both necessary and sufficient to confer efficient translation, even in the absence of a cap or a poly(A) tail (Pestova et al 1996a,b).…”

Section: Introductionmentioning

confidence: 99%

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Translation initiation factor eIF4G1 preferentially binds yeast transcript leaders containing conserved oligo-uridine motifs

Zinshteyn

Rojas-Durán

Gilbert

2017

RNA

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Translational control of gene expression plays essential roles in cellular stress responses and organismal development by enabling rapid, selective, and localized control of protein production. Translational regulation depends on context-dependent differences in the protein output of mRNAs, but the key mRNA features that distinguish efficiently translated mRNAs are largely unknown. Here, we comprehensively determined the RNA-binding preferences of the eukaryotic initiation factor 4G (eIF4G) to assess whether this core translation initiation factor has intrinsic sequence preferences that may contribute to preferential translation of specific mRNAs. We identified a simple RNA sequence motif-oligo-uridine-that mediates high-affinity binding to eIF4G in vitro. Oligo(U) motifs occur naturally in the transcript leader (TL) of hundreds of yeast genes, and mRNAs with unstructured oligo(U) motifs were enriched in immunoprecipitations against eIF4G. Ribosome profiling following depletion of eIF4G in vivo showed preferentially reduced translation of mRNAs with long TLs, including those that contain oligo(U). Finally, TL oligo(U) elements are enriched in genes with regulatory roles and are conserved between yeast species, consistent with an important cellular function. Taken together, our results demonstrate RNA sequence preferences for a general initiation factor, which cells potentially exploit for translational control of specific mRNAs.

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Section: Discussionmentioning

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Translation initiation factor eIF4G1 preferentially binds yeast transcript leaders containing conserved oligo-uridine motifs

Zinshteyn

Rojas-Durán

Gilbert

2017

RNA

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“…The situation with full-length eIF4G and full-length mRNA is likely to be very different because fulllength eIF4G contains two or three RNA-binding sites. The affinity of each binding site to single-stranded RNA is in the low micromolar range (40,41), but the affinity of full-length eIF4G to RNA is about 100-fold higher (K d Х 50 nM) (42). Thus, a combination of the cap-binding activity of eIF4E and the RNAbinding activity of eIF4G would likely stabilize a complex of eIF4E, eIF4G, and mRNA.…”

Section: Discussionmentioning

confidence: 99%

Kinetic Mechanism for Assembly of the m7GpppG·eIF4E·eIF4G Complex

Slepenkov

Korneeva

Rhoads

2008

Journal of Biological Chemistry

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Interaction of the mRNA cap with the translational machinery is a critical and early step in the initiation of protein synthesis. To better understand this process, we determined kinetic constants for the interaction of m 7 GpppG with human eIF4E by stopped-flow fluorescence quenching in the presence of a 90-amino acid fragment of human eIF4G that contains the eIF4E-binding domain (eIF4G(557-646)). The values obtained, k on ‫؍‬ 179 ؋ 10 6 M ؊1 s ؊1 and k off ‫؍‬ 79 s ؊1 , were the same as reported previously in the absence of an eIF4G-derived peptide. We also used surface plasmon resonance to determine kinetic constants for the binding of eIF4E to eIF4G(557-646), both in the presence and absence of m 7 GpppG. The results indicated that eIF4G(557-646) binds eIF4E and eIF4E⅐m 7 GpppG at the same rate, with k on ‫؍‬ 3 ؋ 10 6 M ؊1 s ؊1 and k off ‫؍‬ 0.01 s ؊1 . Our data represent the first full kinetic description of the interaction of eIF4E with its two specific ligands. The results demonstrate that the formation of the m 7 GpppG⅐eIF4E⅐eIF4G(557-646) complex obeys a sequential, random kinetic mechanism and that there is no preferential pathway for its formation. Thus, even though eIF4G(557-646) binds eIF4E tightly, it does not increase the affinity of eIF4E for m 7 GpppG, as has been claimed in several previous publications. We did, in fact, observe increased binding to m 7 GTP-Sepharose in the presence of eIF4G(557-646), but only with recombinant eIF4E that was prepared from inclusion bodies.Recruitment of mRNA to the 43 S translation initiation complex to form the 48 S initiation complex is a highly regulated process that is rate-limiting for protein synthesis under normal circumstances (in the absence of cellular stress, virus infection, etc.) and requires eIF3, 2 poly (A)-binding protein (PABP), and the eIF4 factors (1, 2). The eIF4 factors consist of: eIF4A, a 46-kDa ATP-dependent RNA helicase; eIF4B, a 70-kDa RNAbinding and -annealing protein that stimulates eIF4A activity; eIF4H, a 25-kDa protein that also stimulates eIF4A; eIF4E, a 25-kDa cap-binding protein; and eIF4G, a 185-kDa protein that specifically binds to and co-localizes all of the other proteins involved in mRNA recruitment on the 40 S subunit. A complex of eIF4G, eIF4E, and eIF4A can be isolated from the ribosomal high-salt wash and is termed eIF4F.A critical step in mRNA recruitment is binding of the cap to eIF4E. This occurs mainly by means of -stacking, H-bonding, and ionic interactions inside the narrow cap-binding slot in the concave surface of the protein (3-5). The binding reaction is electrostatically steered and is accompanied by a partial protonation of the m 7 G moiety at N1 and hydration of the complex (6, 7). The kinetics of cap analog binding to eIF4E have been studied by stopped-flow fluorescence quenching (8 -11). Authors of the earlier studies interpreted their kinetic data as indicating a two-step association reaction, in which an initial fast binding is followed by a slow conformational rearrangement (8, 9). However, subsequen...

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“…In yeast, deletion of RNA1 or RNA3 is ts, when removed from the sole isoform of eIF4G, and further RNA motif removal is lethal (Berset et al 2003). However, singly deleting RNA2 or RNA3 is lethal when combined with the tif4631-459 mutations that impair eIF4E binding (Park et al 2011).…”

Section: Mrna Recruitment Of the 43s Picmentioning

confidence: 99%

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

2016

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In this review, we provide an overview of protein synthesis in the yeast Saccharomyces cerevisiae. The mechanism of protein synthesis is well conserved between yeast and other eukaryotes, and molecular genetic studies in budding yeast have provided critical insights into the fundamental process of translation as well as its regulation. The review focuses on the initiation and elongation phases of protein synthesis with descriptions of the roles of translation initiation and elongation factors that assist the ribosome in binding the messenger RNA (mRNA), selecting the start codon, and synthesizing the polypeptide. We also examine mechanisms of translational control highlighting the mRNA cap-binding proteins and the regulation of GCN4 and CPA1 mRNAs.

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RNA-binding activity of translation initiation factor eIF4G1 from Saccharomyces cerevisiae

Abstract: ABSTRACT

Cited by 64 publications

References 37 publications

Translation initiation factor eIF4G1 preferentially binds yeast transcript leaders containing conserved oligo-uridine motifs

Translation initiation factor eIF4G1 preferentially binds yeast transcript leaders containing conserved oligo-uridine motifs

Kinetic Mechanism for Assembly of the m7GpppG·eIF4E·eIF4G Complex

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

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