Mextli proteins use both canonical bipartite and novel tripartite binding modes to form eIF4E complexes that display differential sensitivity to 4E-BP regulation

Peter, D.; Weber, Ramona; Köne, Carolin; Chung, Min-Yi; Ebertsch, Linda; Truffault, Vincent; Weichenrieder, Oliver; Igreja, Cátia; Izaurralde, Elisa

doi:10.1101/gad.269068.115

Cited by 20 publications

(39 citation statements)

References 24 publications

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“…2), in their free state and in complex with a cap analog m 7 GDP, are similar to those observed for other eIF4Es from diverse organisms (Marcotrigiano et al, 1997;Gross et al, 2003;Monzingo et al, 2007;Paku et al, 2012;Papadopoulos et al, 2014;Peter et al, 2015a;2015b). The Cm eIF4E crystals contain four independent copies in the asymmetric unit, all adopting a crescent-shaped conformation formed by a strongly bent beta sheet composed of eight antiparallel b-strands.…”

Section: Resultssupporting

confidence: 72%

“…Metazoan eIF4E-binding proteins (m4E-BPs) also contain the C motif (Mader et al, 1995;Marcotrigiano et al, 1999) and inhibit translation initiation by competing for the same binding site on the eIF4E surface, thus blocking the assembly of active translation eIF4F complexes (Mader et al, 1995;Matsuo et al, 1997;Marcotrigiano et al, 1999;Gross et al, 2003). In addition to the C motif, m4E-BPs also contain a downstream noncanonical (NC) domain that forms a loop and binds to a highly conserved hydrophobic lateral surface of eIF4E (Mizuno et al, 2008;Gosselin et al, 2011;Paku et al, 2012;Lukhele et al, 2013;Igreja et al, 2014;Peter et al, 2015aPeter et al, , 2015b). An elbow loop downstream of the C motif of m4E-BPs induces the bending of the peptide backbone, thus allowing the NC loop to reach and contact the lateral hydrophobic pocket of eIF4E (Kinkelin et al, 2012;Peter et al, 2015aPeter et al, , 2015b.…”

mentioning

confidence: 99%

“…In addition to the C motif, m4E-BPs also contain a downstream noncanonical (NC) domain that forms a loop and binds to a highly conserved hydrophobic lateral surface of eIF4E (Mizuno et al, 2008;Gosselin et al, 2011;Paku et al, 2012;Lukhele et al, 2013;Igreja et al, 2014;Peter et al, 2015aPeter et al, , 2015b). An elbow loop downstream of the C motif of m4E-BPs induces the bending of the peptide backbone, thus allowing the NC loop to reach and contact the lateral hydrophobic pocket of eIF4E (Kinkelin et al, 2012;Peter et al, 2015aPeter et al, , 2015b. In plants, only lipoxygenase 2 (LOX2) and the beta subunit of the nascent polypeptide-associated complex (BTF3) have been identified as putative eIF4E interacting proteins (Freire et al, 2000;Freire, 2005), and very little is known about their association with eIF4E.…”

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confidence: 99%

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Structure of eIF4E in Complex with an eIF4G Peptide Supports a Universal Bipartite Binding Mode for Protein Translation

et al. 2017

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The association-dissociation of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) with eIF4G is a key control step in eukaryotic translation. The paradigm on the eIF4E-eIF4G interaction states that eIF4G binds to the dorsal surface of eIF4E through a single canonical alpha-helical motif, while metazoan eIF4E-binding proteins (m4E-BPs) advantageously compete against eIF4G via bimodal interactions involving this canonical motif and a second noncanonical motif of the eIF4E surface. Metazoan eIF4Gs share this extended binding interface with m4E-BPs, with significant implications on the understanding of translation regulation and the design of therapeutic molecules. Here we show the high-resolution structure of melon (Cucumis melo) eIF4E in complex with a melon eIF4G peptide and propose the first eIF4E-eIF4G structural model for plants. Our structural data together with functional analyses demonstrate that plant eIF4G binds to eIF4E through both the canonical and noncanonical motifs, similarly to metazoan eIF4E-eIF4G complexes. As in the case of metazoan eIF4E-eIF4G, this may have very important practical implications, as plant eIF4E-eIF4G is also involved in a significant number of plant diseases. In light of our results, a universal eukaryotic bipartite mode of binding to eIF4E is proposed.The initiation of protein synthesis is a key control and highly regulated step in eukaryotic gene expression (Sonenberg and Hinnebusch, 2009). Translation initiation leads to the assembly of the large (60S) and small (40S) ribosomal subunits into an active 80S ribosome that is able to locate the correct start codon of the mRNA molecule. This is facilitated by the coordinated actions of at least 12 protein initiation factors (Browning, 2004;Jackson et al., 2010;Hinnebusch, 2011;Hinnebusch and Lorsch, 2012; Browning and BaileySerres, 2015). In cap-dependent translation, binding of the eukaryotic translation initiation factor 4F (eIF4F) to the 7-methyl guanosine cap (m 7 G cap) at the 59 end of mRNA drives the attachment of the mRNA to the ribosomal 43S preinitiation complex. In mammals, eIF4F is built by three proteins: the mRNA 59 cap binding protein eIF4E, the RNA helicase eIF4A, and the scaffolding protein eIF4G, which contains binding domains for eIF4E, eIF4A, eIF3, and poly(A)-binding protein (PABP; Marcotrigiano et al., 1997;Pestova et al., 2001;Gross et al., 2003). In plants, purified eIF4F heterodimers contain eIF4E and eIF4G (Hinnebusch and Lorsch, 2012); plant eIF4A appears to be loosely associated and is therefore easily lost during purification (Lax et al., 1986). EIF4G associates with eIF3, recruiting the 40S subunit of the ribosome and initiating scanning of the mRNA in the 59 to 39 direction (Jackson et al., 2010;Park et al., 2011). EIF4G additionally binds simultaneously to eIF4E and PABP, the latter being able to interact with the mRNA poly(A) tail (Aitken and Lorsch, 2012; Browning and BaileySerres, 2015), resulting in a transient circularization of the mRNA.Higher plants contain an addit...

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Section: Resultssupporting

confidence: 72%

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confidence: 99%

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confidence: 99%

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Structure of eIF4E in Complex with an eIF4G Peptide Supports a Universal Bipartite Binding Mode for Protein Translation

et al. 2017

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“…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. This indicates that the interaction between eIF4e and the mRNA is mediated almost exclusively through the m 7 GTP part of the cap-0 structure.…”

Section: Nmr Studies Of the Capped Rnamentioning

confidence: 54%

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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“…S1B). The following 30 residues (which we termed auxiliary sequences) are also well conserved and contain several short motifs that may potentially interact with 4EHP, as was observed previously in the D. melanogaster protein Mextli in complex with eIF4E (Peter et al 2015b).…”

Section: Gyf1/2 Proteins Contain Noncanonical and Auxiliary 4ehp-bindmentioning

confidence: 72%

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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Mextli proteins use both canonical bipartite and novel tripartite binding modes to form eIF4E complexes that display differential sensitivity to 4E-BP regulation

Cited by 20 publications

References 24 publications

Structure of eIF4E in Complex with an eIF4G Peptide Supports a Universal Bipartite Binding Mode for Protein Translation

Structure of eIF4E in Complex with an eIF4G Peptide Supports a Universal Bipartite Binding Mode for Protein Translation

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

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

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