RNA-independent interaction between Paip1 and eIF3. (A) GST pull-down experiments were conducted with GST-Paip1 or GST on HeLa cell extracts. Eluates were subjected to SDS-PAGE. Bands unique to GST-Paip1 (circled) were excised and subjected to mass spectrometry analysis. Identified proteins are indicated next to the analyzed bands. Protein sequence coverage is as follows: eIF3a, 15.4%; eIF3b, 14.4%; eIF3c, 20.2%; PABP, 56.0%; eIF3g, -h, and -i, ∼10%. (B) GST pull-downs were conducted with GST-Paip1 or GST in untreated or micrococcal nuclease-treated HeLa cell extracts. Eluates were subjected to SDS-PAGE and Western blotting (WB) using the indicated antibodies. (C) Immunoprecipitation using anti-eIF3 antibody (eIF3) or preimmune goat serum (IgG) was conducted using a HeLa cell extract. Extract (Input) and eluates (IP) were subjected to SDS-PAGE and Western blotting using the indicated antibodies. The Paip1 isoforms and IgG heavy chain (HC) are indicated. (D) GST pull-downs were conducted with GST or GST-Paip1 together with purified eIF3. Eluates were subjected to SDS-PAGE and Western blotting using the indicated antibodies.

Identification of the eIF3-binding domain of Paip1. (A) Schematic representation of Paip1 fragments used in GST pull-downs and Flag immunoprecipitation. Relative binding was evaluated visually. (B) GST pull-downs were conducted with the indicated GST-Paip1 fragments in HeLa cell extracts. Eluates were subjected to SDS-PAGE and Western blotting (WB) with the indicated antibodies. The GST and GST-tagged p65 proteins are indicated. (C) HeLa cells were transfected with plasmids expressing the indicated Flag-tagged Paip1 fragments or left untransfected (NT). Immunoprecipitations were performed 48 h posttransfection. Eluates were subjected to SDS-PAGE and Western blotting with the indicated antibodies.

Paip1-dependent translation stimulation in vivo. (A) Schematic representation of HA-tagged Paip1 fragments expressed in HeLa cells. (B) Cells were transfected with the indicated pTet-HA-Paip1 plasmids together with constructs expressing the Renilla luciferase and the tTA. Cells were placed in medium containing 0 or 300 ng/ml of Tet. Extracts were subjected to SDS-PAGE and Western blotting (WB) with the indicated antibodies. (C) Renilla luciferase activity was quantified in HeLa cell extracts from panel B and normalized on the total protein level. Relative induction of the luciferase reporter was determined by calculating the ratio of Renilla luciferase activity between induced (without Tet) and repressed (300 ng/ml Tet) expression of the indicated HA-tagged Paip1. Error bars denote the standard error of the mean for three independent experiments. (D) Renilla luciferase mRNA was measured by quantitative RT-PCR from total RNA extracted from duplicate HeLa cells from panel B. Total RNA was DNase-treated and reverse transcribed. Using RT products, Renilla mRNA was quantified and normalized against β-actin mRNA. Luciferase mRNA ratio is calculated by the ΔΔC_T method using the repressed expression (+Tet) for each Paip1 construct as a reference. Error bars denote the standard error of the mean for three independent experiments.

Model for the mechanism of Paip1-mediated translation stimulation. Paip1 stabilizes the interaction between eIF4G and PABP by binding to eIF3, which in turn binds to eIF4G and the 40S ribosome (not shown in the figure). m7G, cap structure.

(A) Structural organization and interactions of PABP with Paip proteins. (B) Structural organization of the human Paip1 gene, mRNAs, and proteins. The p51 isoform is generated by alternative splicing of exon 1B and lacks amino acids 10 to 88. The p45 isoform is generated from a downstream AUG present in exon 2. Primers a, b, and c used for RT-PCR are indicated. (C) HeLa cells were treated with siRNA against Paip1 exon 1B. Total RNA was extracted, and RT-PCR experiments were performed with primers a and b for mRNA encoding p65 and p51 isoforms of Paip1 (lanes 1 and 2) and with primers c and b for mRNA encoding p45 isoform (lanes 3 and 4). Size marker and PCR products for the p65 (670 bp), the p51 (430 bp), and the p45 (220 bp) isoforms are indicated. (D) HeLa cells were treated with siRNA against exon 1B (lane 2, siRNA#1) or exon 5 (lane 3, siRNA#2) of the Paip1 gene or with control siRNA (Ctrl.) (left panel). HeLa cells were transfected with pCDNA3 Flag-tagged constructs expressing the p65, p51, or p45 isoform of Paip1 or left nontransfected (Nt.) (right panel). Extracts were subjected to SDS-PAGE and Western blotting (WB) using the indicated antibodies. Positions of molecular weight markers are indicated on the right.

Identification of Paip1-interacting subunit of eIF3. (A) Purified eIF3 was subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. Membrane was subjected to Ponceau S staining and far-Western blotting using ³²P-labeled HMK-Paip1 p45 as a probe. A duplicate membrane was subjected to Western blotting (WB) with PABP antibody. eIF3 subunits and fragments of eIF3a are indicated. (B) GST pull-downs were conducted on recombinant p51 Paip1 with GST-eIF3b, GST-eIF3e, GST-eIF3f, GST-eIF3g, GST-eIF3h, or GST. Eluates were subjected to SDS-PAGE and Western blotting (WB) using Paip1 or GST antibodies. (C) GST pull-downs were conducted with GST-eIF3b, GST-eIF3e, GST-eIF3f, GST-eIF3g, GST-eIF3h, or GST on a HeLa cell extract. Eluates were subjected to SDS-PAGE and Western blotting (WB) using the indicated antibodies.

Simultaneous binding of Paip1, eIF3, PABP, and eIF4GI. GST pull-downs were conducted with GST-Paip1 under the following conditions: in the presence of eIF4GI N and PABP (A) and of eIF4G M+C and eIF3 (B). Eluates were subjected to SDS-PAGE and Western blotting (WB) with the indicated antibodies. Blots were then stained with Coomassie blue stain (CB).

Effect of eIF3 and PABP on Paip1-dependent translation stimulation in vivo. (A) HeLa cells were transfected with the indicated siRNAs, and siRNA 4E-T inverted (15) was used as a control (Ctrl). At 24 h after siRNA transfection, cells were transfected as described in the legend of Fig. 6B using the pTet-HA-Paip1 p45 construct. Cells were placed in medium containing 0 or 300 ng/ml of Tet. Data are expressed as the percentage of the relative increase in induction in cells transfected with control siRNA (set at 100%). Error bars denote the standard error of the mean of three independent experiments. (B) Renilla luciferase mRNA was measured by quantitative RT-PCR from total RNA extracted from duplicate HeLa cells from panel A. Total RNA was DNase treated and reverse transcribed. By using RT reactions, Renilla mRNA was quantified and normalized against β-actin mRNA. The luciferase mRNA ratio is calculated by the ΔΔC_T method using the repressed expression (+ Tet) for each siRNA as a reference. Error bars denote the standard error of the mean for three independent experiments. (C and D) Extracts from panel A were subjected to SDS-PAGE and Western blotting (WB) with the indicated antibodies.