Crystal structure of a minimal eIF4E–Cup complex reveals a general mechanism of eIF4E regulation in translational repression

Kinkelin, K.; Veith, Katharina; Grünwald, Marlene; Bono, Fulvia

doi:10.1261/rna.033639.112

Cited by 57 publications

(94 citation statements)

References 69 publications

(141 reference statements)

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“…Upon addition of the RNA, we observed clear chemical shift perturbations (CSPs) in the eIF4E resonances that directly report on the specific interaction between the cap binding protein and the capped RNA. The large number of residues that undergo CSPs is in agreement with the enclosure of the cap structure into the core of the eIF4E protein (Matsuo et al 1997;Kinkelin et al 2012). In agreement with the limited (Tomoo et al 2005;Liu et al 2009;Peter et al 2015) show that the position of the first nucleotide of the RNA body (pink) is not well defined.…”

Section: Nmr Studies Of the Capped Rnasupporting

confidence: 66%

“…Previously, the affinity between the Dm. eIF4E and m 7 GDP (an RNA cap analog that lacks the complete RNA body and that contains two instead of three phosphate groups) was determined to be ∼700 nM, using ITC (Kinkelin et al 2012). The slightly better affinity of the protein for capped RNA in comparison to that for m 7 GDP suggests that there are a small number of contacts between the cap binding protein and the mRNA that involve parts of the RNA that are outside the methylated guanine and first two phosphates in the cap linker.…”

Section: Figure 4 (A)mentioning

confidence: 99%

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A general method for rapid and cost-efficient large-scale production of 5′ capped RNA

Fuchs

Neu

Sprangers

2016

RNA

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The eukaryotic mRNA 5 ′ cap structure is indispensible for pre-mRNA processing, mRNA export, translation initiation, and mRNA stability. Despite this importance, structural and biophysical studies that involve capped RNA are challenging and rare due to the lack of a general method to prepare mRNA in sufficient quantities. Here, we show that the vaccinia capping enzyme can be used to produce capped RNA in the amounts that are required for large-scale structural studies. We have therefore designed an efficient expression and purification protocol for the vaccinia capping enzyme. Using this approach, the reaction scale can be increased in a cost-efficient manner, where the yields of the capped RNA solely depend on the amount of available uncapped RNA target. Using a large number of RNA substrates, we show that the efficiency of the capping reaction is largely independent of the sequence, length, and secondary structure of the RNA, which makes our approach generally applicable. We demonstrate that the capped RNA can be directly used for quantitative biophysical studies, including fluorescence anisotropy and highresolution NMR spectroscopy. In combination with 13 C-methyl-labeled S-adenosyl methionine, the methyl groups in the RNA can be labeled for methyl TROSY NMR spectroscopy. Finally, we show that our approach can produce both cap-0 and cap-1 RNA in high amounts. In summary, we here introduce a general and straightforward method that opens new means for structural and functional studies of proteins and enzymes in complex with capped RNA.

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Section: Nmr Studies Of the Capped Rnasupporting

confidence: 66%

Section: Figure 4 (A)mentioning

confidence: 99%

A general method for rapid and cost-efficient large-scale production of 5′ capped RNA

Fuchs

Neu

Sprangers

2016

RNA

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“…Both structures show that P 79 brings about a sharp turn in the C-terminal of 4E-BP1 and that 80 GVS/T 82 forms a short antiparallel β-interaction with eIF4E. More recently, this binding interface was also found in homologs of the eIF4E/4E-BP complex from Drosophila melanogaster (36,40), suggesting that the interaction between eIF4E and the nonconsensus motif is widespread among eIF4E binding proteins.…”

Section: Discussionmentioning

confidence: 91%

Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1

Sekiyama

Arthanari

Papadopoulos

et al. 2015

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

105

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The eIF4E-binding protein (4E-BP) is a phosphorylation-dependent regulator of protein synthesis. The nonphosphorylated or minimally phosphorylated form binds translation initiation factor 4E (eIF4E), preventing binding of eIF4G and the recruitment of the small ribosomal subunit. Signaling events stimulate serial phosphorylation of 4E-BP, primarily by mammalian target of rapamycin complex 1 (mTORC1) at residues T 37 /T 46 , followed by T 70 and S 65 . Hyperphosphorylated 4E-BP dissociates from eIF4E, allowing eIF4E to interact with eIF4G and translation initiation to resume. Because overexpression of eIF4E is linked to cellular transformation, 4E-BP is a tumor suppressor, and up-regulation of its activity is a goal of interest for cancer therapy. A recently discovered small molecule, eIF4E/eIF4G interaction inhibitor 1 (4EGI-1), disrupts the eIF4E/eIF4G interaction and promotes binding of 4E-BP1 to eIF4E. Structures of 14-to 16-residue 4E-BP fragments bound to eIF4E contain the eIF4E consensus binding motif, 54 YXXXXLΦ 60 (motif 1) but lack known phosphorylation sites. We report here a 2.1-Å crystal structure of mouse eIF4E in complex with m 7 GTP and with a fragment of human 4E-BP1, extended C-terminally from the consensus-binding motif (4E-BP1 50-84 ). The extension, which includes a proline-turn-helix segment (motif 2) followed by a loop of irregular structure, reveals the location of two phosphorylation sites (S 65 and T 70 ). Our major finding is that the C-terminal extension (motif 3) is critical to 4E-BP1-mediated cell cycle arrest and that it partially overlaps with the binding site of 4EGI-1. The binding of 4E-BP1 and 4EGI-1 to eIF4E is therefore not mutually exclusive, and both ligands contribute to shift the equilibrium toward the inhibition of translation initiation.translation initiation | phosphorylation | protein-protein interaction inhibitor | structure

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“…eIF4G and the 4E-BPs share a conserved, canonical (C) 4E-binding motif with the sequence YX 4 LΦ (where Y, X, L, and Φ represent Tyr, any amino acid, Leu, and a hydrophobic residue, respectively), which binds to the dorsal surface of eIF4E opposite to the cap-binding pocket (Matsuo et al 1997;Marcotrigiano et al 1999;Gross et al 2003). Both eIF4G and the 4E-BPs also contain variable noncanonical (NC) 4E-binding motifs that bind to an eIF4E hydrophobic lateral surface, increasing the affinity of the interaction (Kinkelin et al 2012;Paku et al 2012;Lukhele et al 2013;Igreja et al 2014;Peter et al 2015a,b;Sekiyama et al 2015;Grüner et al 2016). Because eIF4G and 4E-BPs bind to the same surfaces on eIF4E, their binding is mutually exclusive, resulting in translation activation and inhibition, respectively.…”

mentioning

confidence: 99%

GIGYF1/2 proteins use auxiliary sequences to selectively bind to 4EHP and repress target mRNA expression

et al. 2017

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The eIF4E homologous protein (4EHP) is thought to repress translation by competing with eIF4E for binding to the 5 ′ cap structure of specific mRNAs to which it is recruited through interactions with various proteins, including the GRB10-interacting GYF (glycine-tyrosine-phenylalanine domain) proteins 1 and 2 (GIGYF1/2). Despite its similarity to eIF4E, 4EHP does not interact with eIF4G and therefore fails to initiate translation. In contrast to eIF4G, GIGYF1/2 bind selectively to 4EHP but not eIF4E. Here, we present crystal structures of the 4EHP-binding regions of GIGYF1 and GIGYF2 in complex with 4EHP, which reveal the molecular basis for the selectivity of the GIGYF1/2 proteins for 4EHP. Complementation assays in a GIGYF1/2-null cell line using structure-based mutants indicate that 4EHP requires interactions with GIGYF1/2 to down-regulate target mRNA expression. Our studies provide structural insights into the assembly of 4EHP-GIGYF1/2 repressor complexes and reveal that rather than merely facilitating 4EHP recruitment to transcripts, GIGYF1/2 proteins are required for repressive activity.

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Crystal structure of a minimal eIF4E–Cup complex reveals a general mechanism of eIF4E regulation in translational repression

Cited by 57 publications

References 69 publications

A general method for rapid and cost-efficient large-scale production of 5′ capped RNA

A general method for rapid and cost-efficient large-scale production of 5′ capped RNA

Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1

GIGYF1/2 proteins use auxiliary sequences to selectively bind to 4EHP and repress target mRNA expression

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