1. Volume 7, Issue 1, Pages 205-216 (January 2001)
Translational Repression by a Novel Partner of Human Poly(A) Binding Protein, Paip2 
Kianoush Khaleghpour, Yuri V Svitkin, Andrew W Craig, Christine T DeMaria, Rahul C Deo, Stephen K Burley, Nahum Sonenberg 
Molecular Cell 
Volume 7, Issue 1, Pages (January 2001)
DOI: /S (01)00168-X

2. Figure 1
Interaction of Paip2 with PABP and Immunodetection of Paip2 in Cells
(A) Interaction of Paip2 with PABP. GST and GST-Paip2 proteins (0.1 μg) were resolved by SDS–15% PAGE and electroblotted onto a nitrocellulose membrane. The blot was probed with 32P-labeled FLAG-HMK-PABP for Far Western analysis, as described in Experimental Procedures.
(B) Specificity of the Paip2 antiserum. HeLa extract (50 μg) was subjected to immunoblotting. Probing was done with preimmune serum; Paip2 antiserum; Paip2 antiserum preadsorped with GST (2.5 μg); Paip2 antibody preadsorped with GST-Paip2 (2.5 μg); or donkey anti-rabbit horseradish peroxidase–conjugated IgG (2°Ab) on individual Western blot strips. Blots were then probed with a mouse monoclonal anti-actin antiserum to normalize for protein loading.
(C) Identification of the 26 kDa protein. Total HeLa cell extract (100 μg) was subjected to Western blotting using a rabbit polyclonal anti-Paip2 antibody. Immunoprecipitation of HeLa cell extract (1 mg) was performed as described in Experimental Procedures and analyzed by Far Western blotting using 32P-labeled FLAG-HMK-PABP as a probe. Ten percent of the input (lane 1), proteins immunoprecipitated by preimmune serum (lane 2), or by the anti-Paip2 immune serum (lane 3) were resolved by SDS–12.5% PAGE.
(D) Interaction of Paip2 with PABP in vivo. Immunoprecipitation of HeLa cell extract (1 mg) was performed as described in Experimental Procedures and analyzed by Western blotting using rabbit polyclonal anti-Paip2 and anti-PABP antibodies. Ten percent of the input (lane 1), proteins immunoprecipitated by preimmune serum (lane 2), or by the anti-Paip2 immune serum (lane 3) were resolved by SDS–12.5% PAGE.
(E) Analysis of Paip2 in cells. Immunoblot analysis of the total cell extract (50 μg) from cell lines or wheat germ [35S]methionine-labeled Paip2 in vitro translation product. Translation in a micrococcal nuclease–treated wheat germ cell-free system was performed according to the manufacturer's instructions (Promega). The position of Paip2 is indicated.
Molecular Cell 2001 7, DOI: ( /S (01)00168-X)

3. Figure 2 Subcellular Localization of Paip2
HeLa cells were transfected with plasmid-encoding HA-tagged Paip2 (pACTAG-2-Paip2).
(A) Phase contrast.
(B) DAPI staining.
(C) Immunolocalization of Paip2. Indirect immunofluorescence was performed as described in Experimental Procedures using a monoclonal anti-HA antibody (1:1000) and Texas red–conjugated goat anti-mouse IgG antibody (1:400).
(D) Far Western analysis of subcellular fractions. HeLa cells were fractionated as described (Peranen et al. 1990) with the following modification: upon isolation of the nucleoplasmic fraction, the nuclear pellet was not treated with micrococcal nuclease and instead was boiled in 2× Laemmli sample buffer. The postnuclear fraction is obtained from an initial low NP-40-containing wash of the nuclear fraction, whereas the nucleoplasmic fraction is obtained from a high NP-40 wash. Proteins were resolved by SDS–15% PAGE, and electroblotted onto a nitrocellulose membrane; equal volumes were used. The blot was probed with 32P-labeled FLAG-HMK-PABP as described in Experimental Procedures.
Molecular Cell 2001 7, DOI: ( /S (01)00168-X)

4. Figure 3
Paip2 Inhibits Cap-Dependent and Cap-Independent Translation In Vitro
Translation (A, B, C, E, and F) and stability (D and G) of luciferase mRNA. Krebs-2 cell-free translation reactions (12.5 μl) containing unlabeled amino acids and RNasin (400 U/ml; Promega) were programmed with 25 ng of capped (A–D) or 50 ng of CV-IRES containing (E–G) poly(A)+ or poly(A)− mRNA in the presence of the indicated amounts of GST-Paip2 at 30°C for 60 min, as described previously (Svitkin et al. 1984). Following incubation, 3 μl aliquots were assayed for luciferase activity using the luciferase assay kit (Boehringer Mannheim) in a Lumat LB 9507 bioluminometer (EG&G Berthold).
(A) Capped mRNA translation is shown as the average of three independent measurements in a single experiment.
(B) The results of (A) are shown as relative luciferase activity (% of control) with standard deviation from the mean.
(C) Restoration of translation by PABP. The indicated amounts of His-PABP and a constant amount of GST-Paip2 (0.075 μg) were added to Krebs-2 translation reactions, which were programmed with capped poly(A)+ luciferase mRNA (25 ng) under standard conditions. Luciferase activity is shown as the average of two independent assays.
(D) Capped luciferase mRNA stability. Translation reactions (75 μl) were programmed with capped poly(A)+ or poly(A)− luciferase mRNA in the presence or absence of GST-Paip2 (0.9 μg). Aliquots (12.5 μl) were withdrawn from the reaction mixtures at the indicated times and treated with Trizol reagent (Gibco-BRL) to isolate the RNA, which was resolved by formaldehyde–agarose gel electrophoresis, followed by Northern blotting using a 32P-labeled luciferase probe. Relative amounts of recovered mRNA are indicated at the bottom, and values obtained for time 0 were set as 100%.
(E) Translation efficiency of CV-IRES mRNA is shown as the average of one experiment carried out in triplicate.
(F) CV-IRES mRNA translation (% of control) is shown as an average of five independent determinations performed in two independent experiments; standard deviations from the mean values are also shown.
(G) Effect of Paip2 on the stability of CV-IRES luciferase mRNA. Krebs-2 translation reactions were programmed with CV-IRES poly(A)+ or poly(A)− luciferase mRNA under standard conditions. mRNA stability was determined as described in (D).
(H) Effect of Paip2 on translation of capped poly(A)+ bicistronic CAT-HCV IRES-luc mRNA. CAT expression was analyzed by an enzyme-linked immunosorbent assay (Roche). CAT and luciferase activity is an average of three independent determinations; standard deviations from the mean values are also shown. Polyadenylation was performed as described in Experimental Procedures, except that reactions contained cold ATP and the incubation time was 5 min.
Molecular Cell 2001 7, DOI: ( /S (01)00168-X)

5. Figure 4 Paip2 Inhibits 80S Ribosome Initiation Complex Formation
mRNA–ribosome binding was performed in a cycloheximide (0.6 mM)–supplemented Krebs-2 cell extract with 0.15 μg (105 cpm) of 3′ end labeled native α- and β- rabbit globin mRNA (Pestova et al. 1998a). The extract (37.5 μl) was incubated in the absence (control) or presence of 12 μg/ml of GST-Paip2 at 30°C for 15 min, diluted with ice-cold high salt buffer, and analyzed on a 5 ml 15%–30% linear sucrose gradient essentially as described previously (Svitkin et al. 1996). Inhibition of ribosome binding by a cap analog, m7GDP (0.6 mM), is also shown. Values are normalized to the amount of total radioactivity recovered from the gradient. The percentage of mRNA bound to 80S monosomes in the absence or presence of GST-Paip2 was 10.5% and 2.1%, respectively.
Molecular Cell 2001 7, DOI: ( /S (01)00168-X)

6. Figure 5 Paip2 Inhibits Translation In Vivo
HeLa cells (grown to 60% confluence in six-well plates) were infected with vaccinia virus vTF-3 to express T7 RNA polymerase (Fuerst et al. 1986) for 1 hr. Cells were then transfected with increasing amounts of pcDNA3-Paip2 DNA and a reporter pGEM-CAT-HCV IRES-luc DNA (0.5 μg) using Lipofectin (12 μl; Gibco-BRL) according to the manufacturer's instructions. The total amount of transfected DNA was adjusted to 10.5 μg with pcDNA3. Cells were harvested 16 hr posttransfection, and assayed for CAT and luciferase activities (Roche and Promega, respectively). Results are an average of six independent assays with standard deviations from the mean.
Molecular Cell 2001 7, DOI: ( /S (01)00168-X)

7. Figure 6 Mutually Exclusive Binding of Paip1 and Paip2 to PABP
(A) Alignment of homology regions between Paip1 and Paip2.
(B) In vitro competition between Paip1 and Paip2 for PABP binding. Assays were performed in buffer A. His-PABP (0.26 μg) was mixed with 1 μg of competitor protein (first incubation) in 20 μl of buffer A for 10 min at 30°C. The mixture was added to 10 μl of Sepharose resin (coupled to GST, lanes 2 and 3; GST-Paip1, lanes 4–13; or GST-Paip2, lanes 14–23), and incubated at 4°C for 1 hr (second incubation). Unbound His-PABP fractions were removed and dissolved in 2× Laemmli sample buffer. The resin was washed three times with 1 ml of buffer A supplemented with 0.5% Nonidet P-40. Bound proteins were eluted with 2× Laemmli sample buffer. Samples were resolved on an SDS–12.5% polyacrylamide gel, electroblotted onto a nitrocellulose membrane, and probed with a rabbit polyclonal anti-PABP antibody. B and UB designate bound and unbound fractions, respectively.
(C) In vivo competition between Paip1 and Paip2 for PABP binding. HeLa cells were transfected with 5 μg of HA-tagged Paip2 (pACTAG-2-Paip2) or FLAG-tagged Paip1 (pcDNA3-FLAG-Paip1) using Lipofectamine (GIBCO BRL) according to the manufacturer's recommendations. Total HeLa cell extract (400 μg) was subjected to immunoprecipitation using mouse monoclonal anti-HA antibody as described in Experimental Procedures, and analyzed by Western blotting using rabbit polyclonal PABP antibody (upper panel), mouse monoclonal anti-FLAG antibody (middle panel), and mouse monoclonal anti-HAII antibody (lower panel). Ten percent of the material used for immunoprecipitation was loaded directly onto the gel (lanes 1–3). Proteins were immunoprecipitated by anti-HA antibody (lanes 4–6).
Molecular Cell 2001 7, DOI: ( /S (01)00168-X)

8. Figure 7
Paip2-Mediated Inhibition of PABP Binding to A25 and Disruption of the Poly(A)-Organizing Activity of PABP
(A) Binding of hPABP to A25 RNA in the presence of GST-Paip2 was analyzed by an EMSA as described in Experimental Procedures. hPABP (1–190; 40 nM) was incubated in a reaction buffer at 30°C for 10 min in the presence of increasing concentrations (100, 200, and 1000 nM) of the indicated proteins and further incubated with A25 as described in Experimental Procedures. Binding assays were performed in triplicate and the average percentage of probe shifted was determined.
(B) Inhibition of PABP binding to poly(A) RNA by Paip2. The filter binding assay was performed as described in Experimental Procedures with 3000 cpm of A25 RNA (0.01 nM). The percentage of radiolabeled A25 RNA retained on the filter is shown as a function of PABP concentration in the absence (square) or presence (diamond) of GST-Paip2 (100 nM).
(C) The poly(A)-organizing activity of PABP was assayed in the presence of the indicated concentrations of Paip2 as described in Experimental Procedures. Luciferase RNA containing radiolabeled poly(A) of approximately 300 nt in length was incubated with His-PABP (wt; 0.3 μg) at 37°C for 30 min in the presence of increasing concentrations of GST-Paip2. Following incubation, the mixture was subjected to limited digestion with micrococcal nuclease in the presence of RNase A and RNase T1. The resulting RNA fragments were analyzed by electrophoresis in a 7 M urea–containing 10% polyacrylamide gel.
(D) A poly(A)–PABP complex was formed at 37°C for 30 min; the indicated amounts of GST-Paip2 were then added, and incubation was continued at 37°C for an additional 10 min.
Molecular Cell 2001 7, DOI: ( /S (01)00168-X)