4E-T colocalizes with decapping factors in P-bodies. (A and B) Transfected 4E-T colocalizes with endogenous Dcp1a. HeLa cells transfected with HA–4E-T (A) or GFP–4E-T (B) were stained with anti-Dcp1a to visualize endogenous Dcp1a. The localization of HA–4E-T was determined by indirect immunofluorescence with anti-HA (Covance) and Alexa Fluor 594 anti–mouse IgG. Dcp1a was revealed with anti-Dcp1a and either Alexa Fluor 488 anti–rabbit IgG (A) or Alexa Fluor 594 anti–mouse IgG (B). The colocalization of HA–4E-T or GFP–4E-T with Dcp1a appears yellow (right). (C) Endogenous 4E-T colocalizes with transfected myc-EYFP-Me31B. HeLa cells were transfected with myc-EYFP-Me31B and colocalization with endogenous 4E-T was performed with anti–4E-T and Texas Red–conjugated anti–rabbit IgG. Colocalization appears yellow (right).

eIF4E requires interaction with 4E-T for localization to P-bodies, whereas the other components of the eIF4F complex are excluded from P-bodies. (A) HeLa cells were transfected with HA-eIF4E wt, HA-eIF4E W73A, or HA-4E-T Y30A. Localization of proteins was studied by indirect immunofluorescence with anti-HA (Covance) and Alexa Fluor 594 anti–mouse IgG. (B and C) HeLa cells were transfected with GFP-Dcp1b and staining of endogenous eIF4GI (B) or eIF4A (C) was detected with anti-eIF4GI and Texas Red conjugated anti–rabbit IgG or with anti-eIF4A and Alexa Fluor 594 anti–mouse IgG. (D) HeLa extract (50 and 100 μg) was resolved by SDS-8% PAGE and a Western blot was performed with rabbit anti–eIF4GI polyclonal antibody. For eIF4A detection, proteins were resolved by SDS-10% PAGE and Western blot was performed with a mouse anti-eIF4A monoclonal antibody.

Effect of cycloheximide and LMB on 4E-T localization in P-bodies. (A) HeLa cells were treated with cycloheximide (CHX; 10 μg/ml) for 30 min, and immunofluorescence staining of 4E-T and Dcp1a were examined as described above. (B) HeLa cell extract (20 μg) treated with cycloheximide (CHX; 10 μg/ml) for 30 and 60 min was resolved by 10% SDS-PAGE and Western blot analysis with rabbit anti–4E-T and anti-Dcp1a, or monoclonal mouse β-actin was performed. (C) HeLa cells were transfected with HA–4E-T. 36 h after transfection, the medium was replaced with fresh medium (control) or medium containing CHX (100 μg/ml), and cells were incubated for 3 h before fixation. Alternatively, media containing LMB (5 ng/ml) was added to cells and incubated for 5 h before fixation. The colocalization of HA–4E-T was determined by indirect immunofluorescence with anti-HA (Covance) and that of Dcp1a with anti-Dcp1a antibody as described above. The colocalization of HA–4E-T and Dcp1a appears yellow.

4E-T inhibits cap-dependent translation in vivo. (A) Schematic presentation of the bicistronic reporter plasmid (pGEM-LUC-POLIRES-CAT). HeLa cells were infected with the vaccinia virus vTF7-3, and transfected transiently with the reporter plasmid and pcDNA3-4E-T constructs. (B, bottom panel) Western blot analysis of the transfected cells was performed with anti-4E-T antibody. Lane 1, pcDNA3; lane 2, pcDNA3-4E-T; lane 3, pcDNA3–4E-T–Y30A. The interaction of the wt and mutant 4E-T protein with eIF4E was examined by Far Western analysis (top panel) using a [³²P]-labeled eIF4E probe. (C) LUC and CAT activity were measured 16 h after transfection. The LUC/CAT activity ratio is expressed as a percentage of the control (pcDNA3) set at 100%. The experiments were performed four times in duplicate.

eIF4E colocalizes with 4E-T, Dcp1a, and Dcp2 in P-bodies. (A) HeLa cells (CCL2) were fixed and the localization of eIF4E was determined by indirect immunofluorescence with mouse anti-eIF4E monoclonal antibody and Alexa Fluor 594 anti–mouse IgG. The localization of 4E-T was examined with anti–4E-T and that of Dcp2 with anti-Dcp2 and Alexa Fluor 488 anti–rabbit IgG. The colocalization of these factors appears yellow in the merged image. (B) The colocalization of endogenous eIF4E with 4E-T and Dcp1a was examined with anti–4E-T and anti-Dcp1a in HeLa S3 as above. The left panel demonstrates endogenous eIF4E localization in HeLa S3. Zoomed images (right panel) display colocalization of endogenous eIF4E and Dcp1a in P-bodies. Some of the more distinct P-bodies are indicated by arrowheads. (C) HeLa cells were transfected with GFP-4E-T and the staining of endogenous eIF4E was performed with anti-eIF4E antibody as described above. (D) The intensity of fluorescence of endogenous eIF4E in P-bodies from GFP–4E-T–transfected cells (from panel C) was compared against the intensity of fluorescence of eIF4E from nontransfected cells along the path, which is indicated by the arrow. The P-bodies along the path of the arrow are circled in the adjacent panel for comparison for better visibility. The signal was allowed to bleach before quantification to avoid a saturated signal. The graphs plot the intensity of fluorescence against the distance (μm) traversed by the arrow. (E) HeLa cells transfected with GFP-4E-T were incubated with Alexa Fluor 594 anti–mouse IgG (M-AF594) without previous incubation with anti-eIF4E antibody to demonstrate the integrity of the green filter.

Depletion of 4E-T from HeLa cells results in disappearance of decapping factors from P-bodies. (A) HeLa cells were transfected with siRNA against 4E-T or control siRNAs (4AIII inverted or 4E-T inverted). 48 h after transfection, cells were split into chamber slides for immunofluorescence analysis [see (C)] and into a 6-cm dish for Western blot analysis. 60 h after transfection, protein cell extracts were prepared and 20 μg of extract was resolved by SDS-10% PAGE. Western blotting was performed with rabbit anti–4E-T and anti-eIF4E antibodies, or mouse anti–β-actin monoclonal antibody (Sigma-Aldrich). 4E-T levels were quantified against β-actin, which served as a loading control, and the level of protein in the negative control (4AIII inverted) transfected cells was set as 100. (B) HeLa extract (20 μg) as in (A) was resolved by 10% SDS-PAGE and Western blotting was performed with rabbit anti-Dcp1a and anti-Xenopus p54 (Xp54) antibodies, or monoclonal mouse anti-eIF4E (SK) and anti–β-actin antibodies. (C) HeLa (CCL2) cells were transfected with control siRNA (4AIII inverted) or 4E-T siRNA and indirect immunofluorescence was performed 60 h after transfection with rabbit anti–4E-T and anti-Dcp1a antibodies, or monoclonal mouse anti-eIF4E antibody. (D) HeLa S3 cells were transfected with 4E-T siRNA or control siRNA (4AIII inverted). 24 h after transfection, cells were transfected with myc-EYFP-Me31B; 36 h later, extracts were collected for SDS-PAGE analysis or fixed for immunofluorescence. Cell extracts were resolved by 10% SDS-PAGE and Western blot analysis with rabbit anti–4E-T, or monoclonal anti-myc (9E10) and anti–β-actin was performed. Indirect immunofluorescence was performed to assess the localization of 4E-T, and the localization of myc-EYFP-Me31B was assessed by direct immunofluorescence. The right most panels show the enlarged bordered image.

siRNA-mediated depletion of 4E-T affects mRNA turnover. HeLa cells were transfected with siRNA against 4E-T or a control (Ctrl) siRNA (4AIII inverted). Protein and RNA were harvested for use in Western (C and D) and Northern (A, B, and E) analysis, respectively. (A and B) HeLa cells were transfected with siRNA against 4E-T or with control siRNA and later cotransfected with pTet-β-globin or pTet-β-globin c-fos or GM-CSF ARE and pTet Off. Cells were treated with doxycycline (1 μg/ml) ∼72 h after transfection to block transcription. Total RNA was isolated at 0.5, 2, 3, and 4 h for β-globin/GM-CSF ARE assays (A) and 0.5, 1.5, 3, and 6 h for β-globin/c-fos ARE assays (B) and analyzed by Northern blot. GAPDH served as a loading control. mRNA half-lives were calculated from Northern blots and normalized against GAPDH levels. (C) Protein extracts were collected at 6 h after treatment with doxycycline (1 μg/ml) from cells that were transfected with the reporter plasmid and pTetOff and resolved by SDS-PAGE. Lanes 1 and 4: glo = β-globin + pTetOff; lanes 2 and 5: fos = β-globin/c-fos ARE + pTetOff; lanes 3 and 6: GM = β-globin/GMCSF ARE + pTetOff. (D) Protein extracts were collected at the latest time point of actinomycin D treatment and resolved by 10% SDS-PAGE. Western blot analysis was done with anti–4E-T and anti–β-actin antibodies. NT = nontransfected. (E) At 48 h after transfection, the half-lives of p21 mRNA were assessed by using actinomycin D (5 μg/ml) for the indicated amount of time. Total RNA (5 μg) was resolved on 1.3% formaldehyde gel and analyzed by Northern blotting. 28S levels and 18S levels served as a loading marker. mRNA half-lives were calculated from Northern blots and normalized against ³²P-labeled 18S levels and plotted on a graph with the zero time point set at 100.