Human Proline-Rich Nuclear Receptor Coregulatory Protein 2 Mediates an Interaction between mRNA Surveillance Machinery and Decapping Complex  Hana Cho, Kyoung Mi Kim, Yoon Ki Kim  Molecular Cell  Volume 33, Issue 1, Pages 75-86 (January 2009) DOI: 10.1016/j.molcel.2008.11.022 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Human Upf1 Interacts with Human Proline-Rich Nuclear Receptor Coregulatory Protein 2 (A) Identification of PNRC1, PNRC2, and Upf2 as Upf1-interacting proteins by yeast two-hybrid screening with a human thymus cDNA library. Yeast strain PBN204 was cotransformed with (1) the GAL4-BD fusion plasmid pBCT or pBCT-Upf1 and (2) GAL4-AD fusion plasmid pACT2 or pACT2-PNRC1, pACT2-PNRC2, or pACT2-Upf2 that was isolated from the positive colonies. Yeast cells were spread on selective media lacking leucine and tryptophan (SD-LW) to select for cotransformants. To select for interacting proteins, isolated cotransformants were then replica-plated onto selective media lacking (1) leucine and tryptophan (SD-LW) for filter assays to allow for the detection of lacZ expression; (2) leucine, tryptophan, and uracil (SD-LWU); and (3) leucine, tryptophan, and adenine (SD-LWA). When two proteins interact with each other, cotransformants will express lacZ, URA3, and Ade2. pBCT-polypyrimidine tract binding protein (PTB) and pACT2-PTB served as the positive control for the protein-protein interaction. pBCT and pACT2 were used as the negative control. (B) Schematic diagrams of Upf1-interacting proteins. Nuclear localization signal (NLS), SH3-binding motif (SH3), NR Box (NR), middle domain of eIF4GI (MIF4G), and the minimal region for Upf1 binding (Upf1) are depicted. Overlying numbers indicate amino acid positions. (C) Immunoprecipitation (IP) of FLAG-Upf1. Cos-7 cells were transiently transfected with pcDNA3-FLAG-Upf1 and pCMV-Myc-PNRC2. Two days after transfection, total proteins were harvested and IP was performed using α-FLAG antibody or mouse IgG (mIgG) as a control. RNase A was added to half of each sample before IP. Western blotting was done to detect the indicated proteins (upper). Cellular protein GAPDH served as a negative control. The three left-most lanes represent 3-fold serial dilutions of total-cell extracts before IP and demonstrate that the western blotting is semiquantitative (upper). Cellular GAPDH mRNA was analyzed using semiquantitative RT-PCR to confirm that RNase A treatment completely removed the cellular RNAs (lower). The three left-most lanes represent 2-fold serial dilutions of RNA and demonstrate that the RT-PCR is semiquantitative. (D) IP of Myc-PNRC2. As in Figure 1C, except that IP was performed using α-Myc antibody and RNase A was not treated. Cellular protein β-actin served as a negative control. (E) In vitro GST pull-down assays. E. coli lysates that express GST, GST-Upf1, GST-Upf1(295-914), or GST-ATE1 were mixed with E. coli lysates that express 6xHis-Xpress-PNRC2. After GST pull-down using glutathione sepharose beads, the purified proteins were analyzed by western blotting (WB) using α-GST antibody (upper) or α-PNRC2 antibody (lower). The locations of molecular weight (MW) markers are indicated on the left. Molecular Cell 2009 33, 75-86DOI: (10.1016/j.molcel.2008.11.022) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Human PNRC2 Interacts with Human Dcp1a (A) Mammalian two-hybrid analyses. HeLa cells were transiently cotransfected with four plasmids: (1) an effector plasmid that expresses GAL4 DNA binding domain only (GAL4-BD; pM) or GAL4-BD fusion protein (pM-Dcp1a or pM-p53), (2) an effector plasmid that expresses herpes simplex virus VP16 transcription activation domain only (VP16-AD; pVP16) or VP16-AD fusion protein (pVP16-PNRC2 or pVP16-SV40 large T), (3) the reporter plasmid pFR-Luc, which encodes firefly luciferase (FLuc) cDNA downstream of a basic transcriptional promoter (TATATA) with five tandem repeats of the yeast GAL4 binding sites, and (4) the reporter plasmid pRL-CMV, which encodes Renilla luciferase (RLuc) cDNA and serves to control for variations in the efficiencies of transfection and protein recovery. One day after transfection, total-cell extracts were harvested. The protein-protein interactions were then assayed by measuring FLuc activity, which was normalized to RLuc activity. Normalized FLuc activity of the cells transfected with pM and pVP16 was set to 1. Each bar and lines represent the mean and standard deviation values of three independently performed transfections. (B) IP of FLAG-Dcp1a. As in Figure 1C, except that Cos-7 cells were transiently cotransfected with either pcDNA3-FLAG or pcDNA3-FLAG-Dcp1a, pCMV-Myc-PNRC2, and pCMV-Myc-Upf1. IP was performed using α-FLAG antibody. Western blotting was performed to detect the indicated proteins (upper). Cellular GAPDH mRNA was analyzed using semiquantitative RT-PCR to confirm that RNase A treatment completely removed the cellular RNAs (lower). (C) In vitro GST pull-down assays. As in Figure 1E, except that E. coli lysates that express GST, GST-PNRC2, GST-Upf1, GST-Upf1(295-914), or GST-ATE1 were mixed with E. coli lysates that express 6xHis-Xpress-Dcp1a. The purified proteins were subjected to western blotting (WB) using α-GST antibody (upper) or α-Xpress antibody (lower). Molecular Cell 2009 33, 75-86DOI: (10.1016/j.molcel.2008.11.022) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Downregulation of PNRC2 Abrogates NMD of Globin mRNA and Glutathione Peroxidase 1 mRNA Harboring PTC and NMD of Natural NMD Substrates (A and B) HeLa cells were transiently transfected with the indicated siRNA. Two days later, cells were retransfected with (1) NMD reporter plasmids pmCMV-Gl and pmCMV-GPx1 that are either PTC-free (Norm) or PTC-containing (Ter) and (2) the phCMV-MUP reference plasmid. (A) Western blotting of Upf1 and PNRC2. β-actin served as a control for variations in protein loading (upper). (B) RT-PCR of Gl mRNA (upper) and GPx1 mRNA (lower). The level of Gl mRNA or GPx1 mRNA was normalized to the level of MUP mRNA. Normalized levels of Norm mRNA in the presence of each siRNA were set to 100%. RT-PCR results obtained in three independent experiments varied by less than 24% for Gl mRNA and 3% for GPx1 mRNA. (C–E) HeLa cells were transiently transfected with the indicated siRNA. Two days later, cells were treated with 100 μg/ml 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) to block transcription. Cells were harvested and total RNAs were purified at the indicated time points. (C) Semiquantitative RT-PCRs of COMMD7, IARS, and GAPDH mRNAs. GAPDH mRNA was used to control for variations in RNA purification and RT-PCR. (D and E) The intensity of each band was quantified, and the levels of COMMD7 mRNA (D) and IARS mRNA (E), which were normalized to GAPDH mRNA, were plotted as a function of time after DRB treatment. Error bars specify the extent of deviation among three independent experiments. (F and G) Immunostaining of PNRC2. HeLa cells were transiently transfected with plasmids expressing HA-PNRC2 (F) or HA-Rck/p54 (G). Two days after transfection, cells were stained with α-Dcp1a antibody and α-HA antibody (F) or α-PNRC2 antibody and α-HA antibody (G). Nucleus was stained with DAPI. Molecular Cell 2009 33, 75-86DOI: (10.1016/j.molcel.2008.11.022) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Tethered PNRC2 Downstream of Normal Termination Codon Elicits mRNA Decay (A) HeLa cells were transiently transfected with the indicated plasmids. Two days after transfection, protein and RNA were purified. The β-6bs mRNA and βUAC-6bs mRNA were analyzed by semiquantitative RT-PCR. The levels of β-6bs mRNA and βUAC-6bs mRNA were normalized to the level of MUP mRNA. The normalized levels of β-6bs mRNA and βUAC-6bs mRNA that were obtained in the presence of pMS2-HA were then set to 100%. (B) As in Figure 4A, except that HeLa cells were cotransfected with the effector plasmids as indicated. (C and D) As in Figures 3A and 3B, except that HeLa cells were transiently transfected with PNRC2 siRNA, Upf1 siRNA, Upf2 siRNA, or a nonspecific Control siRNA. Two days later, cells were retransfected with (1) a test plasmid pcβ-6bs, (2) an effector plasmid expressing MS2, MS2-Upf1, MS2-Upf2, MS2-HA, or MS2-HA-PNRC2, and (3) the reference plasmid phCMV-MUP. The downregulation efficiency was determined by western blotting (upper). The β-6bs mRNA was analyzed using semiquantitative RT-PCR (lower). The mRNA levels obtained in three independent experiments varied by less than 7% (C) or 1% (D). Molecular Cell 2009 33, 75-86DOI: (10.1016/j.molcel.2008.11.022) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 The Hyperphosphorylation of Upf1 Preferentially Increases Its Binding to PNRC2 and Dcp1a and Triggers the Movement of Hyperphosphorylated Upf1 into P Bodies (A) Hyperphosphorylated Upf1 preferentially binds to PNRC2 and Dcp1a. IPs of FLAG-Upf1-WT and FLAG-Upf1-G495R/G497E. As in Figure 1D, except that Cos-7 cells were transiently transfected with pCMV-Myc-PNRC2 and either pcDNA3-FLAG-Upf1-WT or pcDNA3-FLAG-Upf1-G495R/G497E, the latter of which encodes the hyperphosphorylated form of Upf1. IP was performed using α-FLAG antibody or mIgG. Western blotting of IP samples was performed using the indicated antibodies. The levels of coimmunopurified proteins were normalized to the level of immunopurified FLAG-Upf1. The normalized level obtained in IP of FLAG-Upf1-WT was set to 1. (B–D) Immunostaining of FLAG-Upf1-WT (B and D) and FLAG-Upf1-G495R/G497E (C). As in Figures 3F and 3G, except that HeLa cells were transiently transfected with plasmids expressing either FLAG-Upf1-WT (B and D) or FLAG-Upf1-G495R/G497E (C). Five hours before fixing, cells were treated with either DMSO (B and C) or 75 nM OA (D). Molecular Cell 2009 33, 75-86DOI: (10.1016/j.molcel.2008.11.022) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 Downregulation of PNRC2 Abolishes the Movement of Hyperphosphorylated Upf1 into P Bodies and Inhibits the Interaction between Upf1 and Dcp1a (A–D) Immunostaining of FLAG-Upf1-G495R/G497E (A and B) and FLAG-Upf1-WT (C and D). HeLa cells were transfected with control siRNA or PNRC2 siRNA and, 2 days later, retransfected with plasmid expressing FLAG-Upf1-G495R/G497E (A and B) or FLAG-Upf1-WT (C and D). One day later, cells were stained with α-FLAG antibody (A-D) and α-Dcp1a antibody (C and D). Five hours before fixing, cells were treated with 75 nM OA (C and D). (E) IP of Myc-Upf1-WT or Myc-Upf1-G495R/G497E. HeLa cells (2 × 107) were transfected with PNRC2 siRNA or nonspecific control siRNA. Two days after transfection, cells were transiently retransfected with plasmids expressing FLAG-Dcp1a and either Myc-Upf1-WT or Myc-Upf1-G495R/G497E. IP was performed using α-FLAG antibody. The cellular protein GAPDH served as a negative control. Molecular Cell 2009 33, 75-86DOI: (10.1016/j.molcel.2008.11.022) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 A Model Illustrating the Hyperphosphorylation of Upf1 by SMG1 Kinase after PTC Recognition, Its Movement into P Bodies, and Recruitment of the Decapping Complex, Which Are Mediated by the Serial Interactions of Hyperphosphorylated Upf1-PNRC2-Dcp1a-Dcp2 The details are described in the Discussion. P in circle indicates a phosphate group; EJC, exon junction complex; AUG, translation initiation codon; UAA, translation termination codon; PTC, premature termination codon. Molecular Cell 2009 33, 75-86DOI: (10.1016/j.molcel.2008.11.022) Copyright © 2009 Elsevier Inc. Terms and Conditions