Article Figures & Data
Figures
- FIG 1
EB2 is an mRNP cap-associated protein. (A) Top, 100 μg of S7 nuclease-treated cytoplasmic HeLa cell extracts were incubated with an m7GTP cap affinity matrix. Cell extracts were incubated in the absence or presence of m7GpppG cap analog (lanes 3 to 6), and proteins retained on the column were analyzed by Western blotting. Input (lanes 1 and 2) corresponds to 50 μg of cytoplasmic cell extract. Bands shown in each panel correspond to the same Western blot but were cropped for simplicity. Bottom, EB2 protein purified from bacteria was incubated with an m7GTP cap affinity matrix, and proteins bound to the column (lane 4) or from the flowthrough (lane 6) were analyzed by Western blotting. (B and C) HeLa total cell extracts expressing Flag-tagged EB2 were subjected to immunoprecipitation (IP) using an anti-Flag M2 affinity gel in the absence (lanes 3 and 4) or presence (lanes 5 and 6) of RNase A, and associated proteins were analyzed by Western blotting. Input (lanes 1 and 2) corresponds to 1/10 of cell extracts.
- FIG 2
EB2 interacts in vitro with the eIF4G and PABP initiation factors through its C-terminal domain. (A) Schematic representation of both recombinant GST-EB2 fusion proteins and the eIF4G plus eIF4G deletion mutants used. (B) GST pulldown assays were performed using GST, GST-EB2, GST-EB2Nter, or GST-EB2Cter and [35S]methionine-radiolabeled proteins eIF4GNter, eIF4Gp100, PABP, or eIF4E, synthesized in vitro in a rabbit reticulocyte lysate (RRL), under untreated or RNase-treated conditions as indicated. (C) GST pulldown assays were also performed with GST or GST-PABP fusion proteins and [35S]methionine-radiolabeled proteins EB2 (full length), EB2Nter, or EB2Cter, synthesized in vitro in RRL, under untreated or RNase-treated conditions. Eluates were subjected to SDS-PAGE and analyzed by autoradiography. In lane 1, the equivalent of 1/10 of the total RRL used in each assay was loaded onto the gel as an input control.
- FIG 3
Stimulation of mRNA translation is independent of the promoter used to control the expression of the reporter mRNA. (A) Schematic representation of the reporter constructs used. The reporter plasmids carry the Renilla luciferase-coding sequence preceded by the β-globin 5′UTR (glo-RLuc) under the control of the viral BMRF1 or BDLF1 promoter. (B) Quantification by RT-qPCR of the amount of cytoplasmic endogenous BMRF1 and BDLF1 mRNA expressed in HEK293BMLF1-KO cells transfected with an EB1 expression vector together with different amounts of an EB2 expression vector. The Western blot shows the expression of the EB1 and EB2 proteins after transfection of the corresponding expression vectors in HEK293BMLF1-KO cells. Expression of the endogenous BMRF1 protein is also shown. (C and D) Panels a, mRNA quantification by RT-qPCR of the cytoplasmic luciferase-encoding mRNA expressed from the pBMRF1-RLuc (C) and pBDLF1-RLuc (D) reporter plasmids (using GAPDH as an internal control); panels b, total luciferase activity measured at 24 h posttransfection; panels c, translational efficiency calculated by normalizing the total luciferase activity by reference to the amount of cytoplasmic luciferase mRNA. The results are expressed as mean ± standard deviation (SD) from three independent experiments (n = 3). *, P < 0.05; **, P < 0.01 (two-tailed paired t test).
- FIG 4
The RNA-binding protein EB2 stimulates translation of its associated mRNPs in a hybrid system. (A) Schematic representation of the reporter constructs used and the protocol followed. The reporter plasmid carries the Renilla luciferase gene driven by the β-globin 5′UTR (glo-RLuc) under the control of the CMV promoter. HeLa cells were initially transfected or not by the reporter plasmid. The cellular ribosomal fraction was then pelleted by ultracentrifugation at 36 h posttransfection. Part of the ribosomal fraction purified from HeLa cells that had been mock transfected (RH) or transfected with an EB2 expression plasmid (RH-EB2) was transferred to the supernatant of rabbit reticulocyte lysate (RRL) that had been depleted of its ribosomes to generate the in vitro translation hybrid system (51). The other part of the ribosomal fraction was used to extract total mRNA that was quantified by RT-qPCR. This purified naked mRNA fraction (containing the glo-RLuc mRNA) was transferred to the hybrid system, which was supplemented with cellular ribosomes RH or RH-EB2. The mRNP (glo-RLuc mRNP) containing the ribosome fraction was used to directly supplement the RRL depleted of its ribosomes. For both mRNP and mRNA, translation was then carried out for 30 min at 30°C before determination of Renilla activity. (B) EB2 detection by Western blotting using either the HeLa total cytoplasmic fraction (S10) or the ribosomal fraction (RH) or supernatant (S100), both obtained after ultracentrifugation of HeLa S10 through a sucrose cushion. (C) Different amounts (0.5 or 6 μg per 107 cells) of the glo-RLuc plasmid were transfected in HeLa cells in the absence or presence of EB2. At 36 h after transfection, cytoplasmic mRNA was purified and quantified by RT-qPCR. (D) In vitro-transcribed or cytoplasmic mRNA purified from HeLa cells transfected with the glo-RLuc plasmid was translated in the hybrid system containing a HeLa ribosomal fraction either in the absence (RH) or in the presence (RH-EB2) of EB2. (E) Ribosomal pellets containing mRNPs obtained from 6 μg of glo-RLuc or 0.5 μg of glo-RLuc plus EB2 were collected and translated directly in the hybrid system. For panels C to E, results are presented as mean ± SD from three independent experiments (n = 3). **, P < 0.01 (two-tailed paired t test).
- FIG 5
EB2 strongly reduces the sensitivity of its associated mRNPs to translation initiation inhibitors. (A) Schematic representation of mRNA expressed from the constructs containing the Renilla luciferase reporter gene driven by either the 5′UTR of β-globin or the EMCV or HCV IRES. These were in vitro transcribed with a cap and a poly(A) tail in the case of the glo-RLuc construct, without a cap but with a poly(A) tail in the case of the EMCV-RLuc construct, or without a cap and a poly(A) tail for the HCV-RLuc construct. (B) For mRNAs, luciferase activity from 2.7 nM glo-RLuc mRNA translated for 30 min in the hybrid system containing ribosomes prepared from mock-transfected HeLa cells (RH) or cells transfected with an EB2 expression vector (RH-EB2) in the presence of 100 μM cap analog (m7GpppG) (panel a), 10 μM hippuristanol (panel b), or 0.2 μl of RRL expressing L protease (panel c) is shown. The EMCV-RLuc construct was used as a control in panels a and c, while in panel b the HCV-RLuc construct was used as a control. For mRNPs, luciferase activity from mRNPs associated with the ribosome pellet prepared from HeLa cells transfected with 6 μg or 0.5 μg of expression vector for the glo-RLuc reporter, respectively, without (RH) or with (RH-EB2) an expression vector for EB2 is shown. The translation was carried out for 30 min in the hybrid system in the presence of 100 μM cap analog (m7GpppG) (panel a), 10 μM hippuristanol (panel b), or 0.2 μl of RRL expressing L protease (panel c). The efficiency of cleavage of eIF4G by the L protease in both the RH and RH-EB2 lysates was verified by Western blotting (bottom panel). (C) Luciferase activity from mRNP associated with the ribosome pellet prepared from HeLa cells transfected with 6 μg or 0.5 μg of glo-RLuc expression vector, respectively, without (RH) or with (RH-EB2) an expression vector for EB2. Translation was carried out for 30 min in the hybrid system in the presence of different concentrations of puromycin (panel a) or cycloheximide (panel b). The results are expressed relative to the control, which was set to 100%, and they are presented as mean ± SD from three independent experiments (n = 3). *, P < 0.05; **, P < 0.01 (two-tailed paired t test).
- FIG 6
Model for translation initiation of EB2-associated mRNPs. EB2 protein is loaded onto the mRNP during the nuclear steps through its interaction with the CBC. In the cytoplasm, EB2 stabilizes the interaction between eIF4G and PABP and hence contributes to the stabilization of the closed-loop mRNA organization by binding both proteins via its C-terminal domain.
Tables
- TABLE 1
Cellular proteins interacting with EB2a
Category and UniProt accession no. Protein Description Coverage (%) Reference RNA metabolism Q0VCY7 SRSF1 Serine/arginine-rich splicing factor 1 17.34 18 Q3SZR8 SRSF3 Serine/arginine-rich splicing factor 3 23.17 18 Q3TWW8 SRSF6 Serine/arginine-rich splicing factor 6 9.14 Q99020 hnRNPA/B Heterogeneous nuclear ribonucleoprotein A/B 12.98 P52597 hnRNPF Heterogeneous nuclear ribonucleoprotein F 18.31 P31943 hnRNPH Heterogeneous nuclear ribonucleoprotein H 43.43 P02057 hnRNPK Heterogeneous nuclear ribonucleoprotein K 25.29 P52272 hnRNPM Heterogeneous nuclear ribonucleoprotein M 8.77 Q00839 hnRNPU Heterogeneous nuclear ribonucleoprotein U 21.09 Q4R7L5 DDX1 ATP-dependent RNA helicase DDX1 10.41 O00571 DDX3X ATP-dependent RNA helicase DDX3X 32.78 P17844 DDX5 Probable ATP-dependent RNA helicase DDX5 22.31 Q501J6 DDX17 Probable ATP-dependent RNA helicase DDX17 24.15 O75533 SF3B1 Splicing factor 3B subunit 1 3.74 Q13435 SF3B2 Splicing factor 3B subunit 2 2.68 A1A4K8 U2AF35 Splicing factor U2AF 35-kDa subunit 29.11 Q96T37 RBM15 Putative RNA-binding protein 15 3.99 53 P29341 PABPC1 Polyadenylate-binding protein 1 11.64 Other P08775 RBP1 DNA-directed RNA polymerase II subunit RPB1 24.20 Q8TAQ2 SMARCC2 SWI/SNF complex subunit SMARCC2 18.4 P35658 Nup214 Nuclear pore complex protein Nup214 1.67 ↵a Coimmunoprecipitation experiments were conducted with Flag-EB2 expressed in HEK293BMLF1-KO cells. After a first immunoprecipitation using an anti-Flag antibody resin, all samples were treated with RNase A. They were then subjected to a second immunoprecipitation using a specific antibody against EB2. Protein complexes recovered after this second immunoprecipitation were analyzed by MS.
