Anti-proliferative protein Tob negatively regulates CPEB3 target by recruiting Caf1 deadenylase

Abstract

Tob is a member of the anti-proliferative protein family, which functions in transcription and mRNA decay. We have previously demonstrated that Tob is involved in the general mechanism of mRNA decay by mediating mRNA deadenylation through interaction with Caf1 and a general RNA-binding protein, PABPC1. Here, we focus on the role of Tob in the regulation of specific mRNA. We show that Tob binds directly to a sequence-specific RNA-binding protein, cytoplasmic polyadenylation element-binding protein 3 (CPEB3). CPEB3 negatively regulates the expression of a target by accelerating deadenylation and decay of its mRNA, which it achieves by tethering to the mRNA. The carboxyl-terminal RNA-binding domain of CPEB3 binds to the carboxyl-terminal unstructured region of Tob. Tob then binds Caf1 deadenylase and recruits it to CPEB3 to form a ternary complex. The CPEB3-accelerated deadenylation was abrogated by a dominant-negative mutant of either Caf1 or Tob. Together, these results indicate that Tob mediates the recruitment of Caf1 to the target of CPEB3 and elicits deadenylation and decay of the mRNA. Our results provide an explanation of how Tob regulates specific biological processes.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

CPEB directly binds Tob in vitro and in vivo. (A) COS-7 cells were transfected with pHA–CMV5–CPEB3 and either pME–Flag (lanes 1 and 3) or pME–Flag–Tob (lanes 2 and 4). The cell extracts were subjected to an immunoprecipitation assay (IP) using anti-Flag antibody. The immunoprecipitates (lanes 3 and 4) and inputs (lanes 1 and 2, 10% of the amount immunoprecipitated) were analysed by western blotting (WB) using the indicated antibodies. (B) COS-7 cells were transfected with pFlag–CMV5–CPEB4 and either pME–HA (lanes 1 and 3) or pME–HA–Tob (lanes 2 and 4). The cell extracts were subjected to an immunoprecipitation assay (IP) using anti-HA antibody. The immunoprecipitates (lanes 3 and 4) and inputs (lanes 1 and 2, 10% of the amount immunoprecipitated) were analysed as in (A). (C) Rat brain extract was immunoprecipitated (IP) using anti-CPEB3 antibody or, to control for non-specific IP, preimmune serum. The immunoprecipitates (lanes 2 and 3) and input (lane 1, 10% of the amount of immunoprecipitated) were analysed by western blotting using the indicated antibodies. (D) Coomassie-stained gel of purified recombinant GST (lane 1), GST–Tob (1–285) (lane 2), MBP–CPEB3 (lane 3) and MBP–CPEB4 (lane 4). (E) GST (lanes 1 and 2) or GST–Tob (1–285) (lanes 3 and 4) was immobilized on Glutathione Sepharose resin. The resins were incubated with either MBP–CPEB3 (lanes 1 and 3) or MBP–CPEB4 (lanes 2 and 4), and bound proteins were analysed by western blotting with the indicated antibodies. The dotted lines indicate the places where cutting and pasting of the gel images have been made. The gel images are from the same gel. (F) GST or GST–Tob/BTG was immobilized on Glutathione Sepharose resin (lanes 2–6). The resin was incubated with MBP–CPEB3 (lane 1), and bound proteins were analysed by western blotting with the indicated antibodies. GST and GST-fused BTG1, BTG2, BTG3 and Tob are shown by asterisks.

Figure 2

Identification of the CPEB3-binding site in Tob. (A) Purified recombinant GST-fused Tob fragments (lanes 3–12) were mixed with lysate of COS-7 cells expressing Flag–CPEB3 (lane 1) and GST-pull down assays were performed. The bound proteins were detected by western blotting using anti-Flag (upper panel) and anti-GST (lower panel) antibodies. GST and GST-fused Tob fragments are shown by asterisks. The dotted lines indicate the places where cutting and pasting of the gel images have been made. The gel images are from the same gel. (B) Schematic diagram of the GST–Tob fragments with a summary of the Tob–CPEB3 interaction results. The conserved BTG domain consisting of Box A and Box B, and both the primary (PAM2-C) and the cryptic (PAM2-N) PABP-binding sites are indicated as grey boxes. Black boxes represent the region containing CPEB3-binding site in Tob.

Figure 3

Identification of the Tob-binding site in CPEB3. (A) Purified recombinant GST (right panel, lanes 1, 3, 5, 7, 9, 11 and 13) or GST-fused Tob (1–285) (right panel, lanes 2, 4, 6, 8, 10, 12 and 14) was mixed with lysate of COS-7 cells expressing Flag–CPEB3 fragments (left panel), and GST-pull down assays were performed as in Figure 2A (right panel). (B) Schematic diagram of the Flag–CPEB3 fragments with a summary of the Tob–CPEB3 interaction results. Black boxes represent the region containing Tob-binding site in CPEB3.

Figure 4

Tob mediates interaction between CPEB3 and Caf1. COS-7 cells were transfected with expression plasmids encoding HA–CPEB3, Myc–Tob, and Flag–Caf1 in the indicated combinations. The cell extracts were subjected to an immunoprecipitation assay using anti-Flag antibody. The immunoprecipitates (lanes 5–8) and inputs (lanes 1–4, 10% of the amount immunoprecipitated) were analysed by western blotting using the indicated antibodies.

Figure 5

CPEB3 reduces abundance of CAT mRNA with GluR2 3′UTR. (A) Scheme of the reporter constructs: pFlag–CMV5/TO-CAT-GluR2 3′UTR and its control pFlag–CMV5/TO-CAT. (B) COS-7 cells were transiently transfected with the indicated amounts of pHA–CMV5–CPEB3, pEGFP–C1 reference plasmid expressing EGFP and either pFlag–CMV5/TO-CAT (lanes 1–3) or pFlag–CMV5/TO-CAT-GluR2 3′UTR (lanes 4–6). The cell extracts were subjected to western blot analyses with the indicated antibodies. EGFP served as a transfection/loading control. (C) The amount of Flag–CAT protein was measured and plotted against the amount of pHA–CMV5–CPEB3, where the amount of Flag–CAT protein without pHA–CMV5–CPEB3 was defined as 100%. Error bars represent the s.d. of three independent experiments. (D) COS-7 cells were transfected with the indicated amounts of pHA–CMV5–CPEB3, pEGFP–C1, pFlag–CMV5/TO-CAT and pFlag–CMV5/TO-CAT-GluR2 3′UTR. Total RNA was prepared from the cells and Flag–CAT mRNA was detected by Northern blotting. (E) The amount of Flag–CAT mRNA was measured and plotted as in (C).

Figure 6

CPEB3 controls mRNA deadenylation. (A) Schematic representation of the BGG (1–39)-GluR2 3′UTR mRNA. CPEB3 binds to multiple regions throughout the length of the GluR2 3′UTR but the precise sites are not known. (B) Steady-state levels of BGG (1–39)-GluR2 3′UTR mRNA were analysed by Northern blotting (upper panel). T-REx-HeLa cells were transiently transfected with the BGG (1–39)-GluR2 3′UTR reporter plasmid, pCMV–5xFlag–GST–CAT reference plasmid and either pHA–CMV5 (lanes 1 and 3) or pHA–CMV5–CPEB3 (lanes 2 and 4) and treated with 0 (lanes 1 and 2) or 50 ng/ml (lanes 3 and 4) tetracycline for 12 h before harvesting. 5xFlag–GST–CAT mRNA served as a transfection/loading control. Results are representative of three independently performed experiments. (C) T-REx-HeLa cells were co-transfected with the BGG (1–39)-GluR2 3′UTR reporter plasmid, pCMV–5xFlag–GST–CAT reference plasmid and either pHA–CMV5 (lanes 1–5 and 11) or pHA–CMV5–CPEB3 (lanes 6–10 and 12). At 8 h post-transfection, BGG (1–39)-GluR2 3′UTR mRNA was induced to express by treatment with tetracycline for 12 h, and cells were harvested at the specified time after the transcription was shut off. BGG (1–39)-GluR2 3′UTR short mRNA half-lives of 2.4 and 1.6 h were calculated from lanes 1–5 and 6–10, respectively. BGG (1–39)-GluR2 3′UTR long mRNA half-lives of 2.7 and 1.8 h were calculated from lanes 1–5 and 6–10, respectively. To mark the deadenylated (A0) RNA, BGG (1–39)-GluR2 3′UTR mRNA that was induced by treatment with tetracycline for 12 h was digested with RNase H in the presence of oligo(dT) (lanes 11 and 12). 5xFlag–GST–CAT mRNA served as a transfection/loading control. Results are representative of three independently performed experiments. (D) Total cell lysate was analysed by western blotting using anti-HA, anti-CPEB3 or anti-GAPDH. (E) The levels of the long (right panel) and short (left panel) forms of the BGG (1–39)-GluR2 3′UTR mRNA were quantified with the level of the mRNA from 0 h time point defined as 100%. Results are the average of three independently performed experiments.

Figure 7

Tethering CPEB3 to the mRNA accelerates deadenylation. (A) Schematic representation of the Flag–CMV5/TO-BGG (1–39)-MS2bs mRNA, where a cassette comprising eight MS2-binding sites was inserted about 30 nucleotides downstream of the termination codon. (B) T-REx-HeLa cells were co-transfected with the pFlag–CMV5/TO-BGG (1–39)-MS2bs reporter plasmid, pCMV–5xFlag–EGFP reference plasmid and either pMS2–HA (lanes 1–5, 16 and 17), pMS2–HA–CPEB3 (lanes 6–10) or pHA–CMV5–CPEB3 (lanes 11–15). One day later, BGG (1–39)-MS2bs mRNA was induced to express by treatment with tetracycline for 2.5 h, and cells were harvested at the specified time after the transcription was shut off (lanes 1–15). mRNA half-lives of 5.4, 1.8, and 5.9 h were calculated from lanes 1–5, 6–10 and 11–15, respectively. BGG (1–39)-MS2bs mRNA was induced to express by treatment with tetracycline for 12 h to analyse steady-state BGG (1–39)-MS2bs mRNA (lane 16). To mark the deadenylated (A0) RNA, BGG (1–39)-MS2bs mRNA that was induced to express by treatment with tetracycline for 12 h was digested with RNase H in the presence of oligo(dT) (lane 17). 5xFlag–EGFP mRNA served as a loading control (lower panel). Results are representative of three independently performed experiments. (C) Total cell lysate was analysed by western blotting using anti-HA or anti-GAPDH.

Figure 8

CPEB3-accelerated deadenylation is mediated by Caf1 and Tob. (A) T-REx-HeLa cells were co-transfected with the pFlag–CMV5/TO-BGG (1–39)-MS2bs reporter plasmid, pCMV–5xFlag–EGFP reference plasmid, pMS2–HA–CPEB3 (lanes 8–22) and either pCMV–5xMyc–Caf1 D161A (lanes 13–17) or pCMV–5xMyc–Pan2 D1083A (lanes 18–22). As a control, cells were transfected with pMS2–HA and pCMV–5xMyc (lanes 3–7). The transcriptional pulse-chase analysis was performed as described in Figure 7B. mRNA half-lives of 4.9, 1.9, 5.4 and 2.0 h were calculated from lanes 3–7, 8–12, 13–17 and 18–22, respectively. Steady-state BGG (1–39)-MS2bs mRNA (lanes 1 and 23) and the deadenylated (A0) mRNA (lanes 2 and 24) were analysed as in Figure 7B. 5xFlag–EGFP mRNA served as a transfection/loading control. Results are representative of two independently performed experiments. (B) T-REx-HeLa cells were co-transfected with the pFlag–CMV5/TO-BGG (1–39)-MS2bs reporter plasmid, pCMV–5xFlag–EGFP reference plasmid, pMS2–HA–CPEB3 (lanes 8–22), and either pCMV–Myc–Tob (1–160) (lanes 13–17) or pCMV–Myc–Tob (110–218) (lanes 18–22). As a control, cells were transfected with pMS2–HA and pCMV–Myc (lanes 3–7). The transcriptional pulse-chase analysis was performed as described in Figure 7B. mRNA half-lives of 4.3, 2.2, 5.1 and 4.0 h were calculated from lanes 3–7, 8–12, 13–17 and 18–22, respectively. Steady-state BGG (1–39)-MS2bs mRNA (lanes 1 and 23) and the deadenylated (A0) mRNA (lanes 2 and 24) were analysed as in Figure 7B. 5xFlag–EGFP mRNA served as a transfection/loading control. Results are representative of two independently performed experiments. (C, D) Total cell lysate was analysed by western blotting using anti-HA, anti-Myc or anti-GAPDH antibody.

Figure 9

Downregulating Tob or CPEB stabilizes endogenous GluR2 mRNA in SK-N-SH neuroblastoma cells. (A) SK-N-SH neuroblastoma cells were transfected with Tob/Tob2 siRNA (closed triangle), CPEB3/CPEB4 siRNA (closed rectangle) or a control luciferase siRNA (closed circle). Cells were harvested at the specified time after the transcription was shut off using actinomycin D. GluR2 mRNA (left panel) and GAPDH mRNA (right panel) were analysed by real-time PCR. The levels of the mRNAs were quantitated, where the level of the mRNAs from 0 h time point was defined as 100%. Error bars represent the s.d. of three independent experiments. (B) Downregulation of Tob/Tob2 and CPEB3/CPEB4 in SK-N-SH neuroblastoma cells. Tob mRNA, Tob2 mRNA, CPEB3 mRNA, CPEB4 mRNA and 28S rRNA were analysed by real-time PCR. (C) Total RNA that is isolated from HeLa (lane 5) or SK-N-SH (lane 6) cells was analysed by semi-quantitative RT–PCR. GluR2 mRNA, CPEB3 mRNA, CPEB4 mRNA, Tob mRNA, Tob2 mRNA, or 28S rRNA was amplified. The leftmost four lanes, which analysed two-fold dilutions of RNA, show that the conditions used for RT–PCR is semi-quantitative. (D) Total cell lysate that is isolated from HeLa (lane 1), COS-7 (lane 2) or SK-N-SH (lane 3) cells was analysed by western blotting using anti-Tob, anti-Caf1 or anti-GAPDH. (E) Total cell extract that is isolated from SK-N-SH cells was immunoprecipitated (IP) in the presence of RNase I using anti-CPEB3 antibody or to control for non-specific IP, preimmune serum. The immunoprecipitates (lanes 2 and 3) and inputs (lane 1, 10% of the amount of immunoprecipitated) were analysed by western blotting using the indicated antibodies.

Figure 10

Schematic representation of Tob-mediated mRNA decay. (A) Transcript-specific mRNA decay. Tob mediates the recruitment of Caf1 deadenylase to the mRNA bound by the sequence-specific RNA-binding protein CPEB3 to negatively regulates its gene expression. (B) General mRNA decay. Tob generally mediates the recruitment of the Caf1–Ccr4 complex to the poly(A) tail of mRNA bound by the general RNA-binding protein, PABPC1. The termination of translation triggers the recruitment of Tob–Caf1–Ccr4 to the mRNA (Funakoshi et al, 2007).