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. 2014 Mar;42(5):3298-313.
doi: 10.1093/nar/gkt1265. Epub 2013 Dec 13.

Human 4E-T represses translation of bound mRNAs and enhances microRNA-mediated silencing

Anastasiia Kamenska  1 , Wei-Ting Lu, Dorota Kubacka, Helen Broomhead, Nicola Minshall, Martin Bushell, Nancy Standart
Affiliations

Human 4E-T represses translation of bound mRNAs and enhances microRNA-mediated silencing

Anastasiia Kamenska et al. Nucleic Acids Res. 2014 Mar.

Abstract

A key player in translation initiation is eIF4E, the mRNA 5' cap-binding protein. 4E-Transporter (4E-T) is a recently characterized eIF4E-binding protein, which regulates specific mRNAs in several developmental model systems. Here, we first investigated the role of its enrichment in P-bodies and eIF4E-binding in translational regulation in mammalian cells. Identification of the conserved C-terminal sequences that target 4E-T to P-bodies was enabled by comparison of vertebrate proteins with homologues in Drosophila (Cup and CG32016) and Caenorhabditis elegans by sequence and cellular distribution. In tether function assays, 4E-T represses bound mRNA translation, in a manner independent of these localization sequences, or of endogenous P-bodies. Quantitative polymerase chain reaction and northern blot analysis verified that bound mRNA remained intact and polyadenylated. Ectopic 4E-T reduces translation globally in a manner dependent on eIF4E binding its consensus Y30X4L site. In contrast, tethered 4E-T continued to repress translation when eIF4E-binding was prevented by mutagenesis of YX4L, and modestly enhanced the decay of bound mRNA, compared with wild-type 4E-T, mediated by increased binding of CNOT1/7 deadenylase subunits. As depleting 4E-T from HeLa cells increased steady-state translation, in part due to relief of microRNA-mediated silencing, this work demonstrates the conserved yet unconventional mechanism of 4E-T silencing of particular subsets of mRNAs.

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Figures

Figure 1.
Figure 1.
Drosophila CG32016 is closer in sequence and cellular localization to human 4E-T than to Cup. (A) Cartoon of human 4E-T, Drosophila CG32016 (Dm4E-T) and Cup proteins, indicating degree of conservation between N-terminal third, middle third and C-terminal third regions (% identical, and similar residues in brackets according to EMBOSS needle program). Unfilled circle indicates the approximate location of the canonical eIF4E-binding site, and the filled circle that of the Cup homology domain. (B) Fluorescence imaging of HeLa cells transfected with GFP-4E-T, GFP-Cup and GFP-Dm4E-T/CG32016, also stained with antibodies against the P-body marker protein rck/p54, and with DAPI. Scale 10 µm. (C) Phylogeny tree of human (NM_001164501), mouse (BC033410), X. laevis (NM_001093241, C. elegans 4E-T (Spn-2, NM_065508), Drosophila Cup (CG11181, NM_078769) and 4E-T (CG32016; NM_166798) proteins according to phylogeny.fr (44). Branch length is proportional to the number of substitutions per site.
Figure 2.
Figure 2.
The C-terminus of 4E-T promotes its localization in P-bodies. (A) Summary scheme of N- and C-terminal truncated constructs of GFP-4E-T, with table indicating localization of truncated proteins to P-bodies, or to nuclei. Strong localization indicates localization in a large number of foci in 80% of cells or more, weak localization indicates localization in fewer, smaller foci. DN is dominant negative for endogenous P-bodies. (B) Fluorescence imaging of HeLa cells transfected with full-length and indicated truncated versions of GFP-4E-T, also stained with antibodies against the P-body marker protein rck/p54, and with DAPI. Scale, 10 µm. (C) Western blotting analysis of HeLa cells transfected with indicated 4E-T proteins developed with GFP and tubulin antibodies, as loading control.
Figure 3.
Figure 3.
4E-T recruits eIF4E to P-bodies via Y30XXXXLL and its C-terminal localization sequence. (A) 4E-T binds eIF4E in the yeast two hybrid system via YXXXXLL. Growth in –Leu-Trp drop-out media to select for plasmids, and in –Leu-Trp-Ade-His to select for interactions. (B) GFP-Trap co-immunoprecipitation of eIF4E in HEK293 cells with indicated GFP-4E-T proteins analysed by western blotting with 4E-T and eIF4E antibodies. Input and bound proteins were compared. M is a molecular weight protein standard. (C) Fluorescence imaging of untransfected HeLa cells stained with 4E-T and eIF4E antibodies (top row), and of cells transfected with wild-type and mutant GFP-4E-T proteins (2nd to 4th row), also stained with eIF4E antibodies, and with DAPI. Scale bar, 10 µm.
Figure 4.
Figure 4.
4E-T represses translation of tethered mRNA. (A) Schematic cartoon of the tether function assay. (B) Western blot analysis of HEK293 cells transfected with HA/NHA GFP, Pat1b and 4E-T plasmids, developed with HA and tubulin antibodies. (C) Ratios of Renilla and firefly luciferase activities (top) and mRNAs (relative to GAPDH) (bottom) for HA/NHA-GFP, -Pat1b and -4E-T, with the HA results set to 1.
Figure 5.
Figure 5.
4E-T represses translation of tethered mRNA in a P-body and eIF4E-independent manner. (A) NHA-GFP, and NHA-4E-T wild-type and mutant proteins as indicated were transfected into HEK293 cells alongside Renilla-BoxB and control firefly luciferase plasmids. Mutants in P-body localization (B and E), and eIF4E binding (C and F) were assessed. (B and C) The relative levels of luciferase activities. (E and F) The relative levels of luciferase mRNAs, normalized to GAPDH. *P < 0.05; **P < 0.01, ***P < 0.001, t-test. (D) Western blot analysis of transfected cell lysates developed with HA and tubulin antibodies.
Figure 6.
Figure 6.
Tethering the N terminal 1–180 fragment, which binds eIF4E does not repress translation of bound mRNA. (A) GFP-Trap co-immunoprecipitation of eIF4E in HEK293 cells with GFP alone, GFP-4E-T and GFP-4E-T1-180 proteins analysed by western blotting with 4E-T and eIF4E antibodies. Input and bound proteins were compared. M is a molecular weight protein standard. (B–D) NHA-GFP, and -4E-T wild-type, eIF4E-binding mutant (Y30A) and 1–180 proteins as indicated were transfected into HEK293 cells alongside luciferase plasmids, and the level of firefly (B) and Renilla luciferase (C) activities was determined; with panel D showing their relative activities.
Figure 7.
Figure 7.
Tethered 4E-T does not promote deadenylation of bound mRNA, while loss of eIF4E binding results in modest mRNA decay, involving enhanced binding of CNOT1/7 deadenylase subunits. (A) Northern blot analysis of RNA samples from HEK293 cells transfected with tethering plasmids including NHA-GFP, -4E-T and -4E-T proteins mutant in eIF4E-binding (4Emut, YLL → AAA). RNAs were treated (+) or not (−) with oligo(dT) and RNAse H, and the blot was hybridized with a Renilla luciferase probe, and a GAPDH probe as loading control. (B) Phosphoimager quantitation of northern blot shown in A. (C and D) HEK293 cells were co-transfected with GFP, GFP-4E-T or GFP-4Emut (Y30A) and either FLAG-CNOT1 (C) or FLAG-CNOT7 (D) and lysates were immunoprecipitated with GFP-Trap and the input and bound proteins assessed by western blotting with GFP, FLAG and eIF4E antibodies. M is a molecular weight protein standard. Denaturing gradient gel used in C, standard 15% SDS-PAGE in D.
Figure 8.
Figure 8.
Tether function assay with IRES Renilla reporter mRNA. (A) HEK293 cells were transfected with plasmid DNA encoding control Rluc-BoxB mRNA or with HCV Rluc-BoxB mRNA, and firefly luciferase control plasmid alongside HA and NHA-4E-T plasmids. (B) Ratios of Renilla and firefly luciferase activities (left) and mRNAs (relative to GAPDH) (right) for HA/NHA-4E-T, with the HA results set to 1. (C). Western blot of transfected cell lysates developed with ECL and HA and tubulin antibodies.
Figure 9.
Figure 9.
4E-T silencing enhances protein synthesis, including that of miRNA-regulated reporter and cellular mRNAs. (A) 35S methionine/cysteine incorporation. HeLa cells transfected with a nontargeting scrambled siRNA control and 4E-T siRNA were labelled with 35S methionine/cysteine and the amount of radiolabelled protein was quantitated by trichloroacetic acid precipitation. *P < 0.05. (B) 4E-T participates in miRNA-mediated gene silencing. HeLa cells were transfected with control and siRNA against 4E-T, LSm1 and TNRC6A/B. Renilla luciferase reporter mRNA with 0 or 2 let-7 miRNA target sites was transfected alongside firefly luciferase control reporter mRNA 2 days after siRNA knock-down, and luciferase activities were determined. **P < 0.01. (C) siRNA depletion was assessed by western blotting using indicated antibodies with GAPDH as loading control.
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