Weak binding affinity of human 4EHP for mRNA cap analogs

Zuberek, Joanna; Kubacka, Dorota; Jabłonowska, Agnieszka; Jemielity, Jacek; Stępiński, Janusz; Sonenberg, Nahum; Darżynkiewicz, Edward

doi:10.1261/rna.453107

Cited by 65 publications

(78 citation statements)

References 33 publications

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“…In fact, eIF4E3 binds to m 7 GTP with an equal affinity to that reported for eIF4E2 (32), indicating that this atypical cap-binding strategy can be as effective as the use of an aromatic sandwich. eIF4E3 uses multiple distinct regions to form a pocket defined by Trp98, the aliphatic and backbone residues in the S1-S2 loop including Cys52, and the C terminus.…”

Section: Discussionmentioning

confidence: 72%

eIF4E3 acts as a tumor suppressor by utilizing an atypical mode of methyl-7-guanosine cap recognition

Osborne

Volpon

Kornblatt

et al. 2013

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

104

143

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Recognition of the methyl-7-guanosine (m 7 G) cap structure on mRNA is an essential feature of mRNA metabolism and thus gene expression. Eukaryotic translation initiation factor 4E (eIF4E) promotes translation, mRNA export, proliferation, and oncogenic transformation dependent on this cap-binding activity. eIF4E-cap recognition is mediated via complementary charge interactions of the positively charged m 7 G cap between the negative π-electron clouds from two aromatic residues. Here, we demonstrate that a variant subfamily, eIF4E3, specifically binds the m 7 G cap in the absence of an aromatic sandwich, using instead a different spatial arrangement of residues to provide the necessary electrostatic and van der Waals contacts. Contacts are much more extensive between eIF4E3-cap than other family members. Structural analyses of other cap-binding proteins indicate this recognition mode is atypical. We demonstrate that eIF4E3 relies on this cap-binding activity to act as a tumor suppressor, competing with the growthpromoting functions of eIF4E. In fact, reduced eIF4E3 in high eIF4E cancers suggests that eIF4E3 underlies a clinically relevant inhibitory mechanism that is lost in some malignancies. Taken together, there is more structural plasticity in cap recognition than previously thought, and this is physiologically relevant.M etabolism of mRNA is a complex and highly regulated process dependent on the association of the methyl-7-guanosine (m 7 G) cap structure on the 5′ end of transcripts with appropriate proteins (1-3). In mammalian cells, the two major cap-binding proteins are the nuclear cap-binding complex (CBC) (4) and eukaryotic translation initiation factor 4E (eIF4E) (3). Specific cap recognition is key for the fate of transcripts and impacts processes such as mRNA processing and bulk mRNA export via the CBC or the nuclear export of specific transcripts as well as bulk translation by eIF4E (5). NMR and crystallographic studies reveal that m 7 G cap is specifically recognized by both the CBC and eIF4E via intercalation of the m 7 G cap moiety with the side chains of two aromatic residues, i.e., an aromatic sandwich (4, 6-9). This is an electrostatically driven process relying on the partial positive charge of the m 7 G cap and the negative π-electron clouds of the aromatic residues. The aromatic residues are completely conserved in these cap-binding proteins. This recognition motif is used almost exclusively by proteins that specifically bind the m 7 G cap including eIF4E, CBC, and vaccinia virus protein VP39 (4, 10).Dysregulation of cap-binding proteins can have striking physiological consequences. For instance through its cap-binding activity and subsequent effects on gene expression, eIF4E plays an important role in proliferation and survival (1, 3). Indeed, eIF4E is overexpressed in about 30% of human cancers and its overexpression is oncogenic in cell culture and animal models (2, 11). Mutation of the cap-binding site of eIF4E impairs its activities in translation, mRNA export, and oncogenic transform...

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Section: Discussionmentioning

confidence: 72%

eIF4E3 acts as a tumor suppressor by utilizing an atypical mode of methyl-7-guanosine cap recognition

Osborne

Volpon

Kornblatt

et al. 2013

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

104

143

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“…4E-HP, a class II eIF4E, represses caudal and hunchback mRNAs by binding the cap and Bicoid and Nanos/Pum/Brat, respectively, which interact with the mRNAs directly via particular 3Ј-UTR cis-elements (23,24). Subsequent studies of 4E-HP have shown that it binds the m 7 GpppG 30-fold less well than eIF4E1a (27,28). Interestingly, it has also been shown that Argonaute 2, tethered to the target mRNA 3Ј-UTR by micro-RNA (83), also possesses weak cap-binding activity, responsible for repression of translation initiation by micro-RNAs (87).…”

Section: Discussionmentioning

confidence: 99%

“…6). In the case of 4E-T, the wild type protein was compared with a mutant protein, in which Tyr 28 alone or in combination with Leu 33 and Leu 34 residues of the eIF4E-binding site YSKEELL was mutated to alanine to abrogate eIF4E binding (57). As shown in Fig.…”

Section: Xeif4e1b Is Cytoplasmic and Its Expression Is Limited To Oocmentioning

confidence: 99%

“…Thus caudal and hunchback mRNAs, which establish opposing morphogen gradients in Drosophila embryos, are repressed by Bicoid and the Nos-Pum-Brat NRE complex, respectively, in conjunction with d4E-HP, a class II eIF4E, which binds the cap but not eIF4G (23)(24)(25)(26). Intriguingly, recent studies indicate that 4E-HP has a considerably lower affinity for the cap than eIF4E1a (27,28). Given that Drosophila possesses seven genes encoding eight eIF4E-related proteins (26) and that vertebrates contain 4 -6 such proteins, spread among three distinct classes (25,29), which vary in their abilities to interact with the cap, eIF4G, and the 4E-BP family of proteins (which regulate general translation by competing for eIF4G using the shared eIF4E-binding consensus YXXXXL) (25,26), it seems likely that additional noncanonical eIF4E proteins will be found that regulate translation.…”

mentioning

confidence: 98%

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CPEB Interacts with an Ovary-specific eIF4E and 4E-T in Early Xenopus Oocytes

Minshall

Reiter

Weil

et al. 2007

Journal of Biological Chemistry

170

240

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CPEB (cytoplasmic polyadenylation element-binding protein) is an important regulator of translation in oocytes and neurons. Although previous studies of CPEB in late Xenopus oocytes involve the eIF4E-binding protein maskin as the key factor for the repression of maternal mRNA, a second mechanism must exist, since maskin is absent earlier in oogenesis. Using co-immunoprecipitation and gel filtration assays, we show that CPEB specifically interacts, via protein/protein interactions, with the RNA helicase Xp54, the RNA-binding proteins P100(Pat1) and RAP55, the eIF4E-binding protein 4E-T, and an eIF4E protein. Remarkably, these CPEB complex proteins have been characterized, in one or more organism, as P-body, maternal, or neuronal granule components. We do not detect interactions with eIF4E1a, the canonical cap-binding factor, eIF4G, or eIF4A or with proteins expressed late in oogenesis, including maskin, PARN, and 4E-BP1. The eIF4E protein was identified as eIF4E1b, a close homolog of eIF4E1a, whose expression is restricted to oocytes and early embryos. Although eIF4E1b possesses all residues required for cap and eIF4G binding, it binds m 7 GTP weakly, and in pull-down assays, rather than binding eIF4G, it binds 4E-T, in a manner independent of the consensus eIF4E-binding site, YSKEELL. Wild type and Y-A mutant 4E-T (which binds eIF4E1b but not eIF4E1a), when tethered to a reporter mRNA, represses its translation in a cap-dependent manner, and injection of eIF4E1b antibody accelerates meiotic maturation. Altogether, our data suggest that CPEB, partnered with several highly conserved RNA-binding partners, inhibits protein synthesis in oocytes using a novel pairing of 4E-T and eIF4E1b.Selective protein synthesis in oocytes, eggs, and early embryos of many organisms drives several critical aspects of early development, including meiotic maturation and entry into mitosis, establishment of embryonic axes, and cell fate determination. Protein synthesis is usually regulated at the initiation stage, mediated by the 5Ј m 7 GpppN mRNA cap structure bound by the translation initiation complex eIF4F, composed of eIF4E, the cap-binding protein, the RNA helicase eIF4A, and the large scaffold protein eIF4G, which has a consensus binding site YXXXXL for eIF4E, and additional sites for eIF3 and the poly(A)-binding protein. eIF3 recruits the small ribosomal subunit, whereas the eIF4E-eIF4G-poly(A)-binding protein relay results in the so-called "closed loop" model, responsible for the synergistic enhancement of translation by capped and polyadenylated mRNAs. A hallmark of translational control mechanisms is the role of mRNA-binding proteins, which recognize specific (and usually) 3Ј-UTR 2 cis-elements and influence the recruitment of the small ribosomal subunit to the 5Ј cap (reviewed in Refs. 1-3). Probably, the best studied mRNA-binding protein is CPEB1 (cytoplasmic polyadenylation-binding protein 1), characterized in flies, worms, clams, Aplysia, Xenopus, and mammals, which in its conserved C terminus contains two tandem RNA reco...

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“…Like eIF4E, 4EHP binds to 4E-T, but in sharp contrast to eIF4E, it does not associate with eIF4G (18,21). The 4EHP protein has a 30-to 100-fold weaker affinity for the cap than eIF4E due to a twoamino acid substitution in its cap-binding pocket (22).…”

mentioning

confidence: 98%

Cap-binding protein 4EHP effects translation silencing by microRNAs

Chapat

Jafarnejad

Matta‐Camacho

et al. 2017

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

111

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MicroRNAs (miRNAs) play critical roles in a broad variety of biological processes by inhibiting translation initiation and by destabilizing target mRNAs. The CCR4-NOT complex effects miRNA-mediated silencing, at least in part through interactions with 4E-T (eIF4E transporter) protein, but the precise mechanism is unknown. Here we show that the cap-binding eIF4E-homologous protein 4EHP is an integral component of the miRNA-mediated silencing machinery. We demonstrate that the cap-binding activity of 4EHP contributes to the translational silencing by miRNAs through the CCR4-NOT complex. Our results show that 4EHP competes with eIF4E for binding to 4E-T, and this interaction increases the affinity of 4EHP for the cap. We propose a model wherein the 4E-T/4EHP interaction engenders a closed-loop mRNA conformation that blocks translational initiation of miRNA targets.M icroRNAs (miRNAs) are short, ∼22-nucleotide noncoding RNAs that affect gene expression in most eukaryotes. miRNAs mediate posttranscriptional silencing by guiding the miRNA-induced silencing complex (miRISC), an assembly of Argonautes and GW182/TNRC6 proteins, to target mRNAs. Target recognition initiates a succession of events: mRNA translational repression, deadenylation, and mRNA decay (1). miRNAs impair the function of eIF4F, a three-subunit complex composed of eIF4E, the m 7 GTP (cap)-interacting factor; eIF4G, a scaffolding protein; and eIF4A, a DEAD-box RNA helicase (2-5). The silencing activity of miRISC is mediated by the CCR4-NOT deadenylase complex through the scaffolding subunit, CNOT1 (6-8). CNOT1 recruits the DDX6 and 4E-T (eIF4E transporter, also known as EIF4ENIF1) proteins, which are important for miRNA-mediated silencing (9-16). The 4E-T protein is a conserved eIF4E-binding protein, which directly binds to the dorsal surface of eIF4E through its canonical eIF4E-binding YX 4 LL (Y 30 TKEELL) motif and impairs the eIF4E/eIF4G interaction and translation initiation (17). The 4E-T protein also facilitates the decay of CCR4-NOT-targeted mRNAs by linking the 3′-terminal mRNA decay machinery to the cap via its interaction with eIF4E (13).In mammals, eIF4E is the best-studied member of a family of proteins composed of eIF4E (eIF4E1), 4EHP (4E-homologous protein; eIF4E2), and eIF4E3. The 4EHP and eIF4E proteins share 28% sequence identity (18,19). The 4EHP protein is ubiquitously expressed, and it is 5-10 times less abundant than eIF4E in a number of mammalian cell lines (18)(19)(20). Like eIF4E, 4EHP binds to 4E-T, but in sharp contrast to eIF4E, it does not associate with eIF4G (18, 21). The 4EHP protein has a 30-to 100-fold weaker affinity for the cap than eIF4E due to a twoamino acid substitution in its cap-binding pocket (22).The 4EHP protein has primarily been documented as a translation repressor. In the Drosophila embryo, 4EHP associates with the RNA binding protein Bicoid to repress caudal mRNA translation (23). Similarly, 4EHP also represses the hunchback mRNA by binding to the nanos repressive element complex, which consists of nanos...

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Weak binding affinity of human 4EHP for mRNA cap analogs

Cited by 65 publications

References 33 publications

eIF4E3 acts as a tumor suppressor by utilizing an atypical mode of methyl-7-guanosine cap recognition

eIF4E3 acts as a tumor suppressor by utilizing an atypical mode of methyl-7-guanosine cap recognition

CPEB Interacts with an Ovary-specific eIF4E and 4E-T in Early Xenopus Oocytes

Cap-binding protein 4EHP effects translation silencing by microRNAs

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