Interactions between PABPC1, poly(A) nucleases, and TOB. (A) GST pull-down assays examining the interactions between PABPC1 and poly(A) nucleases. In vitro-translated and [³⁵S]methionine-labeled human CCR4, CAF1, PAN2, and PAN3 (top panel) were incubated with MagneGST particle-immobilized GST-PABPC1, and the resulting complexes were resolved by SDS-PAGE followed by Coomassie blue staining (bottom panel) for equal bait loading and then autoradiography (middle panel). PABPC1 and Paip2 were used as positive controls, whereas luciferase (Luc) was used as a negative control. (B) CCR4, CAF1, and TOB (top panel) were in vitro translated, [³⁵S]methionine labeled, and incubated in combinations with MagneGST particle-immobilized GST-PABPC1. GST pull-down (bottom panel) was performed as described for panel A. Luciferase was used as a negative control.

TOB can form a complex with PABP-poly(A). A gel mobility shift assay was carried out to determine TOB's effect on PABPC1-poly(A) complex formation. ³²P-labeled poly(A)₂₅ RNA was incubated at room temperature alone (lane 1), with 0.68 pmol of purified GST-PABP-RRM1 (lanes 2 and 3), or with 0.25 pmol of purified GST-PABPC1 (lanes 6 and 7), followed by the addition of a molar excess of MBP-TOB2 (1.7 pmol for lanes 3 and 6 and 6.8 pmol for lane 7) as indicated. As a control, the probe was also incubated with MBP-TOB2 alone (1.7 pmol for lane 4 and 6.8 pmol for lane 5). The two arrowheads denote signals for complexes containing poly(A)-PABP and poly(A)-PABP-TOB.

PABP binds PAM2 motifs in TOB. (A) Schematic representations of human TOB and TOB2, showing PAM2. Phenylalanines (F) at positions 139 and 274 in TOB as well as positions 140 and 260 in TOB2 are indicated with asterisks and were mutated to alanines to create the TOB(FF) or TOB2(FF) mutants (mut). (B) GST pull-down assay examining PABPC1-binding sites in TOB proteins. In vitro-translated and [³⁵S]methionine-labeled TOB, TOB(FF), TOB2, and TOB2(FF) (left panel) were incubated with MagneGST particle-immobilized GST-PABP and GST-PABC (top panel). GST pull-down was performed as described for Fig. 1.

TOB2 colocalizes with P-bodies in a cell-cycle-related manner. Immunofluorescence microscopy experiment results show the subcellular distributions of V5-TOB2 and P-bodies marked by GFP-DCP1a in proliferating mouse NIH 3T3 cells. V5-TOB2 was visualized using primary mouse anti-V5 antibody followed by secondary goat anti-mouse Alexa 555 antibody. To distinguish cells in G₂ and M phases from cells in G₁ and S phases, cells were stained for a centromere marker protein, CENP-F, by the rabbit primary antiserum against CENP-F and donkey anti-rabbit Alexa 647 antibody. Cell nuclei were detected by DAPI staining.

PABP-dependent enhancement of deadenylation by TOB. (A to D) Northern blots showing deadenylation and decay of the β-globin (BBB) (A and B) or BBB+PTC (D and D) mRNA in the absence (vector) or presence of ectopically expressed wild-type TOB, mutant TOB(FF), CCR4 and wild-type TOB, or CCR4 and mutant TOB(FF). NIH 3T3 B2A2 cells were transiently cotransfected with the reporter plasmid pTetBBB (A and B) or pTetBBB-PTC (C and D), together with a control plasmid (pSVα-globin/GAPDH) and plasmid(s) encoding wild-type TOB, TOB(FF) mutant, CCR4, their combination, or an empty vector, as indicated underneath each panel. The α-globin/GAPDH mRNA was expressed constitutively and served as an internal standard for transfection efficiency and sample handling. The times given at the top correspond to hours after tetracycline addition. Poly(A)⁻ RNA was prepared in vitro by treating the RNA sample from an early time point with oligo(dT) and RNase H. (E) Western blot analysis of total lysates, showing that ectopically expressed TOB-V5 and HA-CCR4 can be readily detected by anti-V5 or anti-HA antibody and that wild-type and mutant TOB proteins are expressed to a similar level. (F) Deadenylation profiles were drawn on the basis of raw data collected from the corresponding Northern blots in panels A to D. For each panel (A to D), the profile for wild-type TOB (red) was superimposed on that for mutant TOB (green) to illustrate the difference in deadenylation kinetics.

F567 in the PABC domain of PABPC1 is important for interaction with TOB. (A) Schematic representation of human PABPC1. RRM, RNA recognition motif; PABP-2nd-C, the second half of the PABPC1 C-terminal domain, previously known as PABP-C2; PABC, poly(A)-binding protein C-terminal motif. Phenylalanine (F) at position 567 is indicated with an asterisk and was mutated to alanine to create the PABPC1(F) or PABP-2nd-C(F) mutant. (B) GST pull-down assay examining the TOB-binding region in PABPC1. In vitro-translated and [³⁵S]methionine-labeled TOB, TOB2, PABPC1, and Paip2 (left panel) were incubated with MagneGST particle-immobilized GST-PABPC1, GST-PABPC1(F), GST-PABP-2nd-C, and GST-PABP-2nd-C(F) (top panel). GST pull-down was performed as described for Fig. 1.

Interactions between TOB proteins, PABPC1, and poly(A) nucleases. COS7 cells were transfected with plasmids expressing V5-tagged TOB, TOB(FF) mutant, TOB2, TOB2(FF) mutant, or pcDNA6-V5 (as control) (A) or along with plasmid expressing HA-tagged CCR4 (B). RNase A-treated cell extracts were first subjected to immunoprecipitation using anti-V5 antibody conjugated to agarose beads. Immunoprecipitates were then separated by SDS-PAGE and analyzed by Western blotting using various antibodies as indicated to the left. The molecular masses of each protein detected are indicated to the right. In both panels, 5% of total lysate used for immunoprecipitation was included as an input, for comparison.