Weak binding affinity of human 4EHP for mRNA cap analogs

Department of Biophysics, Institute of Experimental Physics, Warsaw University, 02-089 Warsaw, Poland.
RNA (Impact Factor: 4.94). 06/2007; 13(5):691-7. DOI: 10.1261/rna.453107
Source: PubMed


Ribosome recruitment to the majority of eukaryotic mRNAs is facilitated by the interaction of the cap binding protein, eIF4E, with the mRNA 5' cap structure. eIF4E stimulates translation through its interaction with a scaffolding protein, eIF4G, which helps to recruit the ribosome. Metazoans also contain a homolog of eIF4E, termed 4EHP, which binds the cap structure, but not eIF4G, and thus cannot stimulate translation, but it instead inhibits the translation of only one known, and possibly subset mRNAs. To understand why 4EHP does not inhibit general translation, we studied the binding affinity of 4EHP for cap analogs using two methods: fluorescence titration and stopped-flow measurements. We show that 4EHP binds cap analogs m(7)GpppG and m(7)GTP with 30 and 100 lower affinity than eIF4E. Thus, 4EHP cannot compete with eIF4E for binding to the cap structure of most mRNAs.

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    • "Arabidopsis class II eIF4E isoform, nCBP protein, interacts weakly with the cap, with the K AS for m 7 GTP 8.5-fold lower than that of AteIF4E (Table 1). In contrast to Ruud et al. paper [13] we obtained binding affinities of AtnCBP for N(7)-methylated cap analogs similar to those observed for AteIF(iso)4E, with the differences not exceeding 1.5-fold, and to values reported for human 4EHP protein [25]. The most prominent difference that may be responsible for the poor binding of the cap by AtnCBP seems to be the lack of the conserved arginine (Arg183 in AteIF4E, equivalent to Lys162 in mammalian eIF4E), that may interact with the cap's phosphate moiety, which is substituted in AtnCBP with isoleucine (Ile166, conserved in close homologs) (Fig. 1C). "
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    ABSTRACT: The assembly of the ribosome on majority of eukaryotic mRNAs is initiated by the recruitment of eIF4E protein to the mRNA 5′ end cap structure. Flowering plants use two eIF4E isoforms, named eIF4E and eIF(iso)4E, as canonical translation initiation factors and possess a homolog of mammalian 4EHP (or eIF4E-2) termed nCBP. Plants from Brassicaceae family additionally conserve a close paralog of eIF4E which in Arabidopsis thaliana has two copies named eIF4E1b and eIF4E1c. In order to assess the efficiency of plant non-canonical (eIF4E1b/1c and nCBP) and canonical (eIF4E and eIF(iso)4E) eIF4E proteins to bind mRNAs we utilized fluorescence titrations to determine accurate binding affinities of five A.thaliana eIF4E isoforms for a series of cap analogs. We found that eIF4E binds cap analogs from 4-fold to 10-fold stronger than eIF(iso)4E, while binding affinities of nCBP and eIF(iso)4E are comparable. Furthermore, eIF4E1c interacts similarly strongly with the cap as eIF4E, but eIF4E1b binds cap analogs ca. 2-fold weaker than eIF4E1c, regardless of the 95% sequence identity between these two proteins. The use of differentially chemically modified cap analogs in binding studies and a detailed analysis of the obtained homology models gave us insight into the molecular characteristic of varying cap-binding abilities of Arabidopsis eIF4E isoforms.
    Full-text · Article · Nov 2014 · Biochemical and Biophysical Research Communications
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    • "The concentration was determined spectrophotometrically on Jasco V-650 or Carry 100 Bio (Varian) spectrophotometers. Human full-length eIF4E was expressed and purified as described earlier [28]. The final step was ion-exchange chromatography to remove misfolded molecules and aggregates (Supplementary Fig. 1). "
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    ABSTRACT: Initiation is the rate-limiting step during mRNA 5' cap-dependent translation, and thus a target of a strict control in the eukaryotic cell. It is shown here by analytical ultracentrifugation and fluorescence spectroscopy that the affinity of the human translation inhibitor, eIF4E-binding protein (4E-BP1), to the translation initiation factor 4E is significantly higher when eIF4E is bound to the cap. The 4E-BP1 binding stabilizes the active eIF4E conformation and, on the other hand, can facilitate dissociation of eIF4E from the cap. These findings reveal the particular allosteric effects forming a thermodynamic cycle for the cooperative regulation of the translation initiation inhibition.
    Full-text · Article · Nov 2013 · FEBS letters
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    • "And lastly, since eIF4G dramatically enhances eIF4E interaction with the mRNA 5′-cap [63], perhaps Bicoid, GIGYF2 or 4E-T may similarly regulate eIF4E2 cap-binding. Indeed, it is no doubt noteworthy that eIF4E2 (and eIF4E1b), negative regulators of translation and components of RNA-binding complexes (see Introduction), bind the cap less efficiently than eIF4E [16,27], allowing them on the one hand to interact with the cap by virtue of being tethered to particular mRNAs, and to be released from the cap on the other hand in response to cues disassembling the complex, without impeding general protein synthesis, and with the potential to be regulated by binding proteins. "
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    ABSTRACT: In addition to the canonical eIF4E cap-binding protein, eukaryotes have evolved sequence-related variants with distinct features, some of which have been shown to negatively regulate translation of particular mRNAs, but which remain poorly characterised. Mammalian eIF4E proteins have been divided into three classes, with class I representing the canonical cap-binding protein eIF4E1. eIF4E1 binds eIF4G to initiate translation, and other eIF4E-binding proteins such as 4E-BPs and 4E-T prevent this interaction by binding eIF4E1 with the same consensus sequence YX 4Lϕ. We investigate here the interaction of human eIF4E2 (4EHP), a class II eIF4E protein, which binds the cap weakly, with eIF4E-transporter protein, 4E-T. We first show that ratios of eIF4E1:4E-T range from 50:1 to 15:1 in HeLa and HEK293 cells respectively, while those of eIF4E2:4E-T vary from 6:1 to 3:1. We next provide evidence that eIF4E2 binds 4E-T in the yeast two hybrid assay, as well as in pull-down assays and by recruitment to P-bodies in mammalian cells. We also show that while both eIF4E1 and eIF4E2 bind 4E-T via the canonical YX 4Lϕ sequence, nearby downstream sequences also influence eIF4E:4E-T interactions. Indirect immunofluorescence was used to demonstrate that eIF4E2, normally homogeneously localised in the cytoplasm, does not redistribute to stress granules in arsenite-treated cells, nor to P-bodies in Actinomycin D-treated cells, in contrast to eIF4E1. Moreover, eIF4E2 shuttles through nuclei in a Crm1-dependent manner, but in an 4E-T-independent manner, also unlike eIF4E1. Altogether we conclude that while both cap-binding proteins interact with 4E-T, and can be recruited by 4E-T to P-bodies, eIF4E2 functions are likely to be distinct from those of eIF4E1, both in the cytoplasm and nucleus, further extending our understanding of mammalian class I and II cap-binding proteins.
    Full-text · Article · Nov 2013 · PLoS ONE
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