The flip-flop configuration of the PABP-dimer leads to switching of the translation function

Figures

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  Figure 1.. PABP forms a homo-dimer through a direct protein-protein interaction. (A) Size exclusion chromatography was carried out with Flag-PABP proteins. Purified Flag-PABP proteins (4 μM) were injected into a Superdex 200 10/300 GL column (GE healthcare). Upper panel: Gel filtration profile of PABP proteins. The positions of dimeric and monomeric PABPs are depicted by arrowheads. Lower panel: The proteins in each fraction resolved by 10% SDS-PAGE and stained by Coomassie Blue. The bands depicted by asterisk (*) are likely either bacterial proteins co-purified with PABP or fragment(s) of PABP generated during the purification steps. (B, C) The physical interaction between Flag-PABP and GST-PABP protein was investigated by (B) Flag-immunoprecipitation and (C) GST pull-down experiments. Western blot analyses were performed with anti-Flag (to detect Flag-PABP) and anti-GST (to detect GST-PABP) antibodies. (D) Confirmation of PABP–PABP interaction using cells. Flag-immunoprecipitation was performed with Huh7-cells expressing 3x Flag-PABP to detect endogenous PABP with or without treatment of an RNase cyanase. Endogenous PABPs co-precipitated with Flag-PABPs were monitored by Western blotting…

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  Figure 2.. Determination of domains in PABP required for the PABP–PABP interaction. (A) Schematic diagram of Flag-tagged PABP and its variants with deletion mutations. (B) Co-immunoprecipitation of GST-tagged full-length PABP with Flag-tagged PABP variants. Flag-PABP variants and GST-PABP mixtures were incubated and precipitated by a Flag antibody-conjugated resin. The resin-bound proteins were visualized by Western blotting using the indicated antibodies. (C) Interaction between C-terminal domain and C-terminal domain of PABP. Flag-1–2–3–4 (2-371) or Flag-C (372–636) proteins were incubated with GST-C (372–636) proteins and a Flag antibody-conjugated resin. The resin-bound proteins were visualized by Western blotting using the indicated antibodies. (D) Interaction between RRM2-3 (97–299) and RRM1-2–3–4 (2–371) of PABP. GST-2–3 (97–299) protein was incubated with Flag-1–2–3–4 (2–371) or Flag-C (372–636) protein and a Flag antibody-conjugated resin. The resin-bound proteins were visualized by Western blotting using the indicated antibodies. (E) Interaction between RRM2-3 (97–299) and RRM2-3 (97–299) of PABP. GST pull-down assays were performed with GST (lanes 7–10) or GST-2–3 (97–299)…

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  Figure 3.. Poly(A) RNA disrupts PABP–PABP interaction. (A) The effect of poly(A)25 RNA on the PABP–PABP interaction was examined by Flag-immunoprecipitation in the presence of increasing amounts of poly(A)25 RNA. Flag-PABP and GST-PABP proteins were incubated together with a Flag resin, and unbound proteins washed away once. Increasing amounts of poly(A)25 RNAs up to the concentration of total PABP proteins (represented as 1 x) were added to the mixtures and incubated again. The resin-bound proteins were visualized by Western blotting using the indicated antibodies. (B) The effect of poly(A)25 RNA on the interaction between GST-C and Flag-C was examined by Flag-immunoprecipitation in the presence of increasing amounts of poly(A)25 RNA similarly to panel (A). (C) The effect of poly(A)25 RNA on the interaction between GST-C and Flag-PABP was examined by Flag-immunoprecipitation in the presence of increasing amounts of poly(A)25 RNA similarly to panel (A).

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  Figure 4.. Mutations of four glutamates (Es) to alanines (As) in the linker2 region increase the RNA-free PABP–PABP interaction. (A) Sequence alignment of linker2 region of PABP homologs from diverse organisms. Four negatively-charged glutamates (E178, E180, E182, E189) in the linker2 region were mutated to alanines. (B) Homo-dimerization of PABP 4EA mutant protein. Size exclusion chromatography was performed with Flag-PABP 4EA mutant proteins as described in Figure 1A. Upper panel: Gel filtration profile of 4EA mutant PABPs. The positions of dimeric and monomeric PABPs are depicted by arrowheads. Lower panel: The proteins in each fraction resolved by 10% SDS-PAGE and stained by Coomassie Blue. The bands depicted by asterisk (*) are likely either bacterial proteins co-purified with PABP or fragment(s) of PABP generated during the purification steps. (C) The interaction between 4EA mutant proteins was greatly increased compared with the interaction between PABP WTs. Co-immunoprecipitation experiments were performed with GST-PABP WT and Flag-PABP WT pair or with GST-PABP 4EA mutant and Flag-PABP 4EA mutant pair using a flag antibody-conjugated resin to isolate wild type PABP (lane 7) and 4EA…

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  Figure 5.. Protein-protein interaction between eIF4G and PABP WT or 4EA mutant. (A) The interaction between eIF4G and RNA-free PABPs. Flag-PABP WT or Flag-PABP 4EA proteins were incubated with a Flag resin, and then incubated with His-eIF4GN proteins. His-eIF4GN proteins associated with Flag-PABP WT (lane 5) or Flag-PABP 4EA proteins (lane 6) were visualized by Western blotting with an anti-eIF4G antibody. (B) The interaction between eIF4G and poly(A)-bound PABPs. Poly(A) pull-down experiments were performed with His-eIF4GN proteins and either Flag-PABP WT (lane 7) or Flag-PABP 4EA (lane 8) proteins. Resin bound proteins were visualized by Western blotting with the indicated antibodies.

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  Figure 6.. Transition from PABP homo-dimer to poly(A)–PABP complex is hampered by increased PABP–PABP homo-dimer interaction. (A) Dissociation of the PABP 4EA-PABP 4EA homo-dimer by poly(A)25 RNA. Flag-immunoprecipitation experiments were performed similarly to Figure 3A, except that Flag-PABP 4EA and GST-PABP 4EA were used instead of Flag-PABP WT and GST-PABP WT in these experiments. Resin bound proteins were visualized by Western blotting with the indicated antibodies. (B) PABP 4EA has a lower binding affinity than PABP WT to poly(A)25 RNA. Electrophoretic mobility shift assays were performed with radiolabeled [32P]-poly(A)25 RNA (1 nM) and serially increasing amounts of Flag-PABP WT or Flag-PABP 4EA mutant proteins in the range from 125 pM to 256 nM. (Left panel) A representative image of 6% native acrylamide gel showing an upper-shift of radiolabeled poly(A)25 probes through associations with PABP proteins. The asterisks (*) depict the supershift bands of radiolabeled poly(A)25 probes at high concentrations of PABP WT and 4EA proteins. (Right panel) The graph shows the radioactivities remained at the protein-free poly(A)25 RNA band. The binding affinity (KD value) was calculated with…

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  Figure 7.. Proper transition from a PABP homo-dimer to a poly(A)–PABP complex is required for translational activation function of PABP. (A) Depletion of endogenous PABP from rabbit reticulocyte lysates was confirmed by Western blotting of control and PABP-depleted lysates with an anti-PABP antibody. Actin was used as a control protein. (B) In vitro translation assays were performed using the PABP-depleted lysates supplemented with various amounts of PABP WT or PABP 4EA proteins from 0 ng to 100 ng for 60 min. In vitro transcription was executed by T7 RNA polymerase with a template DNA containing the Renilla luciferase gene followed by poly(A)120 sequence to produce 5′ capped and 3′ poly(A)-tailed mRNAs. Relative translational efficiencies were calculated by normalizing translational efficiencies of the reporter mRNAs with various amounts of PABP WT or PABP 4EA to that in PABP-undepleted control lysates which was set to 1. The relative translational efficiencies are depicted by the bar graphs. The white columns denoted as undepleted and dPABP represent relative translation efficiencies of PABP-undepleted control and PABP-depleted lysates, respectively. The black and hatched columns…