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Molecular Cell, Volume 33

Supplemental Data

Human Proline-Rich Nuclear Receptor Coregulatory

Protein 2 Mediates an Interaction between mRNA

Surveillance Machinery and Decapping Complex Hana Cho, Kyoung Mi Kim, and Yoon Ki Kim Supplemental Experimental Procedures Plasmid constructions Plasmids pCMV-SPORT6-PNRC1 and pCNS-PNRC2 containing full-length human PNRC1 cDNA (NM_006813) and PNRC2 cDNA (NM_017761), respectively, were purchased from 21C Human Gene Bank, Genome Research Center, KRIBB, Korea.

To construct plasmid pCMV-Myc-PNRC2, which encodes a Myc tag and full-length human PNRC2 cDNA, the BglII/KpnI fragment from pCMV-Myc (Clontech) was ligated to a PCR fragment that contains human PNRC2 cDNA and was digested with BglII and KpnI. PNRC2 cDNA was amplified using pCNS-PNRC2 and two oligonucleotides: 5’-GAAGATCTTCATGGGTGGTGGAGAGAGGTATAACATTC-3’ (sense) and 5’-GGGGTACCCCTTATACCTGTACTTTAAGTAAGGTTTTAAG-3’ (antisense), where the underlined nucleotides specify the BglII and KpnI sites, respectively. PCR amplification was carried out using the Advantage-HF2 PCR Kit (Clontech).

To construct plasmid pCMV-HA-PNRC2, a SalI/NotI fragment from pCMV-Myc-PNRC2 was ligated to a SalI/NotI fragment from pCMV-HA, which is derived from pEGFP-C1 (Clontech) and encodes a HA tag but not green fluorescent protein (GFP) cDNA.

To construct pMS2-HA-PNRC2, which encodes N-terminal oligomerization-defective MS2 coat protein followed by a HA tag and full-length human PNRC2 cDNA, pMS2-HA-Stau1 (Kim et al., 2005) digested with NheI and NotI was ligated to two fragments: (i) a PCR-amplified fragment MS2-HA that contains the MS2 coat protein-encoding sequence and an HA tag and was digested with NheI and XhoI, and (ii) a PCR-amplified fragment that contains human PNRC2 cDNA and was digested with XhoI and NotI. MS2-HA fragment was amplified using pMS2-HA-Stau1 and two oligonucleotides: 5’- CTAGAGTACTTAATACGACTCACTATAG -3’ (sense) and 5’-CCGCTCGAGAGCGTAGTCTGGAACGTCGTATGGGTAG-3’ (antisense), where the underlined nucleotides specify the XhoI site. PNRC2 cDNA was amplified using pCMX-PNRC2 (a gift from Uwe Borgmeyer) as template and two oligonucleotides: 5’-CCGCTCGAGATGGGTGGTGGAGAGAGGTATAACATTC-3’ (sense) and 5’-ATTTGCGGCCGCTTATACCTGTACTTTAAGTAAGGTTTTAAG-3’ (antisense), where the underlined nucleotides specify the XhoI and NotI sites, respectively.

To construct pVP16-HA-PNRC2, pVP16 (Clontech) digested with BamHI and HindIII

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was ligated to a PCR fragment that contains human PNRC2 cDNA and was digested with BamHI and HindIII. PNRC2 cDNA was amplified using pMS2-HA-PNRC2 and two oligonucleotides: 5’- CGGGATCCCCTCAGCAATCGCAGCAAACTCCGGC -3’ (sense) and 5’-CCCAAGCTTCTCGAGTTATACCTGTACTTTAAGTAAGGTTTTAAG -3’ (antisense), where the underlined nucleotides specify the BamHI and HindIII sites, respectively.

To construct plasmid pM-Dcp1a, pM (Clontech) digested with EcoRI and SalI was ligated to a PCR fragment that contains human Dcp1a cDNA and was digested with EcoRI and SalI. Dcp1a cDNA was amplified using pcDNA3-FLAG-Dcp1a (a kind gift from Jens Lykke-Andersen) and two oligonucleotides: 5’-CGGAATTCCTCGAGGAGGCGCTGAGTCGAGCTGGGCAGGAG-3’ (sense) and 5’-GCGTCGACGGTACCTCATAGGTTGTGGTTGTCTTTGTTCTTG-3’ (antisense), where the underlined nucleotides specify the EcoRI and SalI sites, respectively.

To construct plasmid pcDNA3-FLAG-Upf1, a Klenow-filled HindIII/XbaI fragment from pCMV-Myc-Upf1 (a kind gift from Lynne E. Maquat; Kim et al., 2005) was ligated to the Klenow-filled BamHI fragment from pcDNA3-FLAG (a gift from Didier Poncet).

To construct plasmid pcDNA3-FLAG-Upf1-G495R/G497E, a Klenow-filled HindIII/XbaI fragment from pCMV-Myc-Upf1(G495R/G497E) (a kind gift from Lynne E. Maquat; Isken et al., 2008) was ligated to a Klenow-filled BamHI fragment from pcDNA3-FLAG.

For bacterial production of human PNRC2 that harbors N-terminal 6xHis and Xpress tags, pRSET C-PNRC2 was constructed by ligating the BamHI/HindIII fragment from pRSET C (Invitrogen) to a PCR-amplified fragment digested with BamHI and HindIII. The PCR fragment was amplified using the human PNRC2 cDNA expression vector pCMV-Myc-PNRC2 and two oligonucleotides: 5’- CGGGATCCCGATGGGTGGTGGAGAGAGGTATAACATTC’ (sense) and 5’- CCCAAGCTTCTCGAGTTATACCTGTACTTTAAGTAAGGTTTTAAG -3’ (antisense), where the underlined nucleotides specify the BamHI and HindIII sites, respectively.

For bacterial production of human PNRC2 that lacks N-terminal 13-amino acids and harbors N-terminal GST tag, the BamHI/XhoI fragment from pET-GST (a derivative of pET28a; a gift from Hyun Kyu Song) was ligated to a PCR-amplified fragment digested with BamHI and XhoI. The PCR fragment was amplified using the pREST C-PNRC2 and two oligonucleotides: 5’-CCCCTGGATCCAGAAATGTTAGTAAGAACCAACAACAGC-3’ (sense) and 5’-CCCAAGCTTCTCGAGTTATACCTGTACTTTAAGTAAGGTTTTAAG-3’ (antisense), where the underlined nucleotides specify the BamHI and XhoI sites, respectively.

For bacterial production of human Dcp1a that harbors N-terminal 6xHis and Xpress tags, pRSET A-Dcp1a was constructed by ligating the XhoI/KpnI fragment from pRSET A (Invitrogen) and a PCR fragment that contains human Dcp1a cDNA and was digested with XhoI and KpnI. Dcp1a cDNA was amplified using pcDNA3-FLAG-Dcp1a and two oligonucleotides: 5’-CGGAATTCCTCGAGGAGGCGCTGAGTCGAGCTGGGCAGGAG-3’ (sense) and 5’-GCGTCGACGGTACCTCATAGGTTGTGGTTGTCTTTGTTCTTG-3’ (antisense), where the underlined nucleotides specify the XhoI and KpnI sites, respectively.

To construct pcDNA3-FLAG-PNRC1, the Klenow-filled BamHI fragment from pcDNA3-FLAG was ligated to a Klenow-filled HindIII fragment from pCMV-SPORT6-PNRC1. Yeast two-hybrid screening Yeast two-hybrid screening with human Upf1 was performed twice with the human thymus cDNA activation domain (AD) library. Yeast strain PBN204 (Panbionet Inc.,) was co-

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transformed by the polyethylene glycol-lithium acetate method with two hybrid plasmids: (i) a bait plasmid pBCT-Upf1 that encodes a GAL4 DNA BD-fused Upf1 cDNA and the HIS3 marker gene and (ii) pACT2 plasmid that encodes the human thymus cDNA fused to GAL4 AD and the TRP1 marker gene. In our screening, three different reporter genes, URA3, ADE2, and lacZ, each of which was under the control of different GAL4-binding sites, were used to minimize false positives. First, the transformants were spread on selective media lacking leucine, tryptophan, and uracil and containing 2% glucose (SD-LWU), where the transformants can grow when BD-Upf1 interacts with AD-prey proteins. 132 independent colonies grew on selective media SD-LWU. Second, 89 out of 132 colonies grew on selective media lacking leucine, tryptophan, and adenine and containing 2% glucose (SD-LWA). Third, 88 out of 89 colonies showed a blue color in X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside), which was confirmed by filter assay to allow for the detection of β-galactosidase expression.

To re-confirm the specific interaction of BD-Upf1 and AD-prey proteins, the AD-prey DNAs were amplified by PCR using total DNAs purified from the transformants. The amplified PCR fragment, together with a linearized prey vector, was reintroduced into yeast strain PBN204 expressing BD-Upf1. All transformants were confirmed one more time for the specific interaction by checking the expression of URA3, ADE2, and lacZ. Finally, 32 out of 88 independent colonies were true positives. pBCT-polypyrimidine tract binding protein (PTB) and pACT2-PTB served as the positive control for the protein-protein interaction (Oh et al., 1998). pBCT (Panbionet Inc.,) and pACT2 (Clontech Laboratories, Inc.) were used as the negative control. Mammalian two-hybrid analyses For mammalian two-hybrid assays, HeLa cells were transfected with the indicated plasmids. Twenty-four hours after transfection, cells were harvested and dual luciferase activities were measured using the Luciferase assay kit (Promega). Immunoprecipitation Cos-7 cells (3 x 107) or HeLa cells (1 x 107) were transiently transfected with the indicated plasmids using the calcium phosphate method or Lipofectamine 2000 (Invitrogen), respectively, as previously described (Ishigaki et al., 2001; Kim et al., 2005). For IP, large cell numbers (approximately 2 x 107) were required. Therefore, HeLa cells and Cos-7 cells were used for siRNA-mediated downregulation and IP, respectively. However, the same cell lines (HeLa cells) were used for some experiments including (i) Okadaic acid (OA) treatment followed by IP (Figure S5) and (ii) siRNA-mediated downregulation followed by IP (Figure 6E). Where indicated, to stabilize the hyperphosphorylated form of Upf1, phosphatase inhibitors (0.25 mM Na-o-vanadate and 10 mM NaF) were included in all buffers during IP (Chiu et al., 2003). Western Blotting The following antibodies were used: FLAG (Sigma), Myc (Calbiochem), Upf1, Upf2 (gifts from Dr. Lynne E. Maquat), GST (Amersham Pharmacia Biotech), Xpress (Invitrogen), His (Santa Cruz), HA (Roche), Dcp1a (a gift from Dr. Jens Lykke-Andersen), α-phospho(S/T)Q antibody (Cell Signaling), β-actin (Sigma), and GAPDH (Ab Frontier). Antibody against PNRC2 was raised in rabbits or purchased from Proteintech Group, Inc.

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GST pull-down assay GST pull-down assays were performed using recombinant GST, GST-Upf1, GST-Upf1(295-914), GST-PNRC2, GST-ATE1, 6xHis-Xpress-PNRC2, 6xHis-Xpress-Dcp1a, and 6xHis-GFP. Following incubation of GST-fusion proteins and either 6xHis-Xpress-PNRC2 or 6xHis-Xpress-Dcp1a in 1 ml of incubation buffer [10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10% (v/v) glycerol, 0.1% BSA, 0.1% Triton X-100] at 4°C for 2 hr, samples were incubated with Glutathione Sepharose 4B resin for 1.5 hr. After adsorption, the resin was washed five times with incubation buffer, and resin-bound proteins were resolved by 10% SDS-PAGE, followed by Western blotting. siRNA-mediated downregulation of PNRC2 For downregulation using siRNA, we used human cells, because the complete human sequences are available. HeLa cells were grown in DMEM medium (Lonza) containing 10% fetal bovine serum (Lonza). Transient transfections were performed as previously reported (Kim et al., 2005). In brief, cells were transfected with 100 nM of in vitro-synthesized siRNA (Invitrogen) using Oligofectamine (Invitrogen). Control siRNA, Upf1 and Upf2 siRNA sequences were previously described (Kim et al., 2005). Cellular PNRC2 was downregulated using 5’-r(UUGGAAUUCUAGCUUAUCA)d(TT)-3’. Two days after siRNA transfection, cells were retransfected with a pmCMV-Gl test plasmid (Zhang et al., 1998), either nonsense-free (Norm) or nonsense-containing (39Ter), a pmCMV-GPx1 test plasmid (Moriarty et al., 1998), either Norm or 46Ter, and phCMV-MUP reference plasmid (Belgrader et al., 1994) using Lipofectamine 2000 (Invitrogen).

Alternatively, HeLa cells (2x106) were transiently transfected with 0.3 μg of the reporter plasmid pcDNA3-β-6bs or pcDNA3-βUAC-6bs, 0.1 μg of the reference plasmid phCMV-MUP, and 0.5 μg of one of the following effector plasmids: pMS2-HA, pMS2-HA-PNRC2, pcNMS2, pcNMS2-Upf1, or pcNMS2-Upf2. Two days later, cells were harvested. Total protein was purified from half of the cells using passive lysis buffer (Promega), and total RNA was purified from the other half using TRIzol Reagent (Invitrogen).

For mRNA half-life experiments, cells were transfected with 100 nM of in vitro-synthesized siRNA using Oligofectamine. Three days later, cells were treated with 100 μg/ml of 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB). Then cells were harvested and total RNAs were purified at the indicated time points. Semi-quantitative RT-PCR β-Gl, GPx1, MUP, β-6bs, and βUAC-6bs mRNAs were amplified as previously described (Ishigaki et al., 2001; Kim et al., 2005). Briefly, first strand cDNA was synthesized using M-MuLV Reverse Transcriptase (Fermentas) according to the manufacturer’s protocol using 4 μg of RNA. Semi-quantitative RT-PCR was performed with single-stranded cDNA, specific oligonucleotides, and α-[32P]-dATP (PerkinElmer NEN). Labeled PCR products were electrophoresed in 5% polyacrylamide gel, visualized by PhosphorImaging (BAS-2500; Fuji Photo Film Co.), and then quantitated by Multigauge (Fuji Photo Film Co.). A standard curve of intensity versus RNA amount was prepared using 2-fold serial dilutions of purified RNAs, and then the relative amounts of PCR products were determined from the curve.

Endogenous COMMD7, IARS, or SMG7 mRNA was amplified using oligonucleotides: 5’-GTGAAAAGCCTCCTTCTGG-3’ (sense) and 5’-CTGTAGAACTGAGGCAAGG-3’ (antisense), 5’-AAGATGTTGCCAGAGGACG-3’ (sense) and 5’-

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GCATCCTTCACATACTGTC-3’ (antisense), or 5’-CCA AAGGAGACCATCTGACC-3’ (sense) and 5’-CCTCATCTCGGCTTTCC-3’ (antisense), respectively.

Endogenous PNRC1 mRNA or GAPDH mRNA was amplified using oligonucleotides: 5’-AAAGAAGAAGGTGCGGGCC-3’ (sense) and 5’-TAGAAATGCTGATTTCTTC-3’ (antisense) or 5’-TGGCAAATTCCATGGCACC-3’ (sense) and 5’-AGAGATGATGACCCTTTTG-3’ (antisense), respectively. Quantitative Real-time PCR Real-time PCR analyses were performed using the LightCycler™ system (Roche Diagnostics, Mannheim, Germany). Quantitative real-time PCR analyses were performed with single-stranded cDNA and gene-specific oligonucleotides using the Lightcycler 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany) or Lightcycler 480 Probe Master (Roche Diagnostics GmbH, Mannheim, Germany). Lightcycler PCR conditions were initial denaturation 10 mins at 95°C, followed by 45 cycles of 95°C denaturation for 10 s, 57°C annealing for 10 s, and 72°C elongation for 30 s. The expression levels of mRNAs are the means of three independent experiments. Melting curves of the PCR products were performed for quality control.

Lightcycler 480 SYBR Green I Master and oligonucleotides used in semi-quantitative RT-PCR were used for amplification of GPx1 mRNA and MUP mRNA in Figures S2D, S4B, and S4C. β-6bs mRNA was analyzed using Lightcycler 480 SYBR Green I Master and two oligonucleotides: 5’-AGTTGGTGGTGAGGCCCTGG-3’ (sense) and 5’-ACCAGCACGTTGCCCAGGAG-3’ (antisense). To detect Gl mRNA and MUP mRNA in Figure S2C, Lightcycler 480 Probe Master and gene-specific TapMan probes were used: 5’-(6-Fam)TGATCCCGCGCAGACACTGACCTTCA(BHQ1)-3’ for the detection of Gl mRNA and 5’-(Hex)TTCAGACATCAAGGAAAGGTTTGCACAACT(BHQ1)-3’ for the detection of MUP mRNA. Oligonucleotides used in semi-quantitative RT-PCR were used for amplification of Gl mRNA and MUP mRNA. Immunostaining HeLa cells were fixed with 2% paraformaldehyde (Merck) in PBS for 10 min and permeabilized with 0.5% Triton X-100 in PBS for 10 min. Cells were incubated with blocking buffer (1.5% BSA in PBS) for 1 hour and with primary antibodies for 1 hour.

The primary antibodies [α-Dcp1a antibody (a gift from Jens Lykke-Andersen), α-PNRC2 antibody (Proteintech Group, Inc.), α-FLAG antibody (Sigma), α-Myc antibody (Santa Cruz) and α-HA antibody (Roche)] were detected with fluorescein- and rhodamine-conjugated secondary antibodies (Pierce). Nuclei were stained with DAPI (Biotium). Cells were observed with a ZEISS confocal microscope (LSM510 META). Supplemental Acknowledgements We thank Didier Poncet for providing pcDNA3-FLAG, Uwe Borgmeyer for pCMX-PNRC2, and Stephen K. Burley for pGEX-6p-1+NdeI.

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Supplemental References Belgrader, P., Cheng, J., Zhou, X., Stephenson, L. S., and Maquat, L. E. (1994). Mammalian nonsense codons can be cis effectors of nuclear mRNA half-life. Mol. Cell. Biol. 14, 8219-8228. Chiu, S. Y., Serin, G., Ohara, O., and Maquat, L. E. (2003). Characterization of human Smg5/7a: a protein with similarities to Caenorhabditis elegans SMG5 and SMG7 that functions in the dephosphorylation of Upf1. RNA 9, 77-87. Ishigaki, Y., Li, X., Serin, G., and Maquat, L. E. (2001). Evidence for a pioneer round of mRNA translation: mRNAs subject to nonsense-mediated decay in mammalian cells are bound by CBP80 and CBP20. Cell 106, 607-617. Isken, O., Kim, Y. K., Hosoda, N., Mayeur, G. L., Hershey, J. W., and Maquat, L. E. (2008). Upf1 phosphorylation triggers translational repression during nonsense-mediated mRNA decay. Cell 133, 314-327. Kim, Y. K., Furic, L., Desgroseillers, L., and Maquat, L. E. (2005). Mammalian Staufen1 recruits Upf1 to specific mRNA 3'UTRs so as to elicit mRNA decay. Cell 120, 195-208. Moriarty, P. M., Reddy, C. C., and Maquat, L. E. (1998). Selenium deficiency reduces the abundance of mRNA for Se-dependent glutathione peroxidase 1 by a UGA-dependent mechanism likely to be nonsense codon-mediated decay of cytoplasmic mRNA. Mol. Cell. Biol. 18, 2932-2939. Oh, Y. L., Hahm, B., Kim, Y. K., Lee, H. K., Lee, J. W., Song, O., Tsukiyama-Kohara, K., Kohara, M., Nomoto, A., and Jang, S. K. (1998). Determination of functional domains in polypyrimidine-tract-binding protein. Biochem. J. 331 ( Pt 1), 169-175. Zhang, J., Sun, X., Qian, Y., and Maquat, L. E. (1998). Intron function in the nonsense-mediated decay of beta-globin mRNA: indications that pre-mRNA splicing in the nucleus can influence mRNA translation in the cytoplasm. RNA 4, 801-815.

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Figure S1. 6xHis-GFP does not interact with any tested proteins As in Figures 1E and 2C, except that E. coli lysates that express GST, GST-PNRC2, GST-Upf1, GST-Upf1(295-914), or GST-ATE1 were mixed with partially purified 6xHis-GFP. After GST pull-down using Glutathione Sepharose beads, the purified proteins were analyzed by Western blotting (WB) using α-GST antibody (upper) or α-His antibody (lower). Figure S2. Downregulation of PNRC2 abrogates NMD of Gl mRNA and GPx1 mRNA harboring PTC As in Figures 3A and 3B, except that PNRC2-1 siRNA [5’-r(AGUUGGAAUUCUAGCUUAU)d(TT)-3’], which targets a partially overlapping but distinct sequence of PNRC2 mRNA, was used. (A) Western blotting results indicate that the levels of endogenous Upf1 and PNRC2 were downregulated to 1% and 6% of normal levels, respectively. (B) The semi-quantitative RT-PCRs of Gl mRNA (upper) and GPx1 mRNA (lower). The results showed that downregulation of Upf1 and PNRC2 abrogated NMD of PTC-containing Gl mRNA by 2.8-fold and 2.1-fold, respectively (upper), and NMD of PTC-containing GPx1 mRNA by 3.9-fold or 3.6-fold, respectively (lower). RT-PCR results obtained in at least three independent experiments varied by less than 12%. (C-D) Real-time PCRs of Gl mRNA (C) and GPx1 mRNA (D). As in Figure S2B, except that the level of mRNA was analyzed using real-time PCR. The results showed that downregulation of Upf1 and PNRC2 abrogated the NMD of PTC-containing Gl mRNA by 2.4-fold and 3.1-fold, respectively (C), and the NMD of PTC-containing GPx1 mRNA by 5.5-fold or 4.1-fold, respectively (D). The statistical differences in the results were evaluated by two-tailed, equal-sample variance Student’s t-test. Each P-value is indicated above the bar. Figure S3. Downregulation of PNRC2 abrogates NMDs of endogenous COMMD7 mRNA and IARS mRNA (A-B) HeLa cells were transiently transfected with Upf1 siRNA, PNRC2 siRNA, or a nonspecific Control siRNA. Two days later, cells were harvested and total RNAs were purified. (A) The semi-quantitative RT-PCR of endogenous COMMD7 mRNA. The level of COMMD7 mRNA was normalized to that of endogenous SMG7 mRNA. The normalized level of COMMD7 mRNA in the presence of Control siRNA was set to 100%. RT-PCR results obtained in three independent experiments varied by less than 18%. (B) The semi-quantitative RT-PCR of endogenous IARS mRNA. As in Figure S3A, except that IARS mRNA was analyzed by semi-quantitative RT-PCR. RT-PCR results obtained in three independent experiments varied by less than 1%. Figure S4. Supporting results of tethering experiments (A) Western blotting using α-HA antibody confirmed the expression of MS2-HA or MS2-HA-PNRC2 in Figure 4A. (B) Real-time PCR of β-6bs mRNA. The total RNA obtained in Figure 4B was analyzed using real-time PCR. (C) Real-time PCR of β-6bs mRNA. The total RNA obtained in Figure 4D was analyzed using real-time PCR. The statistical differences of the results were evaluated by two-tailed, equal-sample variance Student’s t-test. Each P-value is indicated above the bar.

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(D and E) Tethered eIF4E had no significant effect on the level of β-6bs mRNA. As in Figure 4D, except that HA-eIF4E was tethered. (D) Western blotting results indicated that the levels of endogenous Upf1 and PNRC2 were downregulated to 4% and 5% of normal levels, respectively. (E) The β-6bs mRNA was analyzed using semi-quantitative RT-PCR. The mRNA levels obtained in three independent experiments varied by less than 6%. Figure S5. Preferential interactions of Upf1 with PNRC2 and Dcp1a depend on phosphorylation status As in Figure 5A, except that HeLa cells were transiently transfected with pCMV-Myc-PNRC2 and FLAG-Upf1-WT. Two days after transfection, cells were treated with DMSO (0 nM OA) or 75 nM OA for 5 h. IP was performed using α-FLAG antibody or mIgG. Western blotting of IP samples was performed using the indicated antibodies. The levels of co-immunopurified proteins were normalized to the level of immunopurified FLAG-Upf1. The normalized level obtained in IP of FLAG-Upf1-WT in the absence of OA was set to 1. β-actin served as a negative control. Results are representative of two independent experiments. Figure S6. Immunostaining of Upf1 and marker proteins for P-bodies (A and B) As in Figures 5B and 5C, except that HeLa cells were transiently transfected with plasmids expressing Myc-Rck/p54 and either FLAG-Upf1-WT (A) or FLAG-Upf1-G495R/G497E (B). (C and D) As in Figures 5B and 5C, except that HeLa cells were transiently transfected with plasmids expressing FLAG-Dcp1a and either Myc-Upf1-WT (C) or Myc-Upf1-G495R/G497E (D). Figure S7. The distribution of FLAG-Dcp1a is not affected by downregulation of PNRC2 (A and B) Immunostaining of FLAG-Dcp1a. As in Figures 6A and 6B, except that, two days after siRNA transfection, HeLa cells were retransfected with plasmid expressing FLAG-Dcp1a. One day later, cells were stained with α-FLAG antibody. (C and D) As in Figures 6A, 6B, S7A and S7B, except that transfected cells were harvested and total-cell extracts were analyzed by Western blotting using α-FLAG antibody. The results showed that downregulation of PNRC2 did not significantly affect the expression levels of FLAG-Upf1-G495R/G497E (C) and FLAG-Dcp1a (D). Figure S8. Downregulation of PNRC1 mRNA by 3-fold does not significantly affect NMD of Gl mRNA and GPx1 mRNA harboring PTC As in Figures 3A and 3B, except that PNRC1 siRNA [5’-r(GGAAAGAGGUUUUAAAAUC)d(TT)-3’] was used. (A) Western blotting (left) and semi-quantitative RT-PCR (right) results indicate that the levels of endogenous Upf1 protein and PNRC1 mRNA were downregulated to 2% and 32% of normal levels, respectively. (B and C) The semi-quantitative RT-PCR of Gl mRNA (B) and GPx1 mRNA (C). RT-PCR results obtained in at least three independent experiments varied by less than 6%. (D) Immunostaining of FLAG-PNRC1 and HA-PNRC2. HeLa cells were transiently transfected with plasmids expressing FLAG-PNRC1 and HA-PNRC2. Two days after transfection, cells were stained with α-FLAG antibody and α-HA antibody. Nuclei were stained with DAPI.

Figure S1

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Gα

-FLA

G

IP

Phospho-FLAG-Upf1WB: α-FLAG

WB: α-phospho(S/T)QFLAG-Upf1

0 75 0 75 OA (nM)

pcDNA3 FLAG Upf1 WT

Myc-PNRC21 9 Myc-PNRC2 / FLAG-Upf1

Phospho FLAG Upf11 6 Phospho-FLAG-Upf1 / FLAG-Upf1

WB: α phospho(S/T)Q

WB: α-Myc

WB: α-β-actin β-actin

Dcp1a / FLAG-Upf1Dcp1a

1 7WB: α-Dcp1a

WB: α β actin β actin

Before IP After IP

Figure S6

FLAG-Upf1-WTMyc-Rck/p54 DAPI MergeA

FLAG-Upf1-G495R/G497E

Myc-Rck/p54 DAPI MergeB

Myc-Upf1-WTFLAG-Dcpla DAPI MergeC

Myc-Upf1-G495R/G497E

FLAG-Dcpla DAPI MergeD

Figure S7

ADAPI MergeFLAG-Dcp1aA

Con

trol s

iRN

AA

DAPI MergeFLAG-Dcp1aB

PNR

C2

siR

NA

FLA

G-U

pf1-

G49

5R/G

497E

trol

pcDNA3-

RC

2

rol

C2

FLA

G-D

cp1a

pcDNA3-

C D

siRNA

Con

tMW(kDa) PN

R

130170

957256

FLAG-Upf1-G495R/G497E

4334

FLAG-Dcpla9572

56

43

siRNA

Con

trPN

RC

MW(kDa)

β-actin β-actin

Figure S8

siRNA

Con

trol

Upf

1PN

RC

1

PNRC1 mRNA

ontro

l

pf1

NR

C1

siRNA

A

SMG7 mRNA

Co

Up

PN

Upf1

siRNA

GAPDH

3 2

100

93±

32± PNRC1 mRNA (%)

BsiRNA

m m m

Con

trol

Upf

1

PNR

C1

siRNA

m m m

Con

trol

Upf

1

PNR

C1

C

Nor

m

Nor

m

Nor

m

Ter

Ter

Ter

Gl mRNA

MUP mRNA

0 1 100

7±0

0 ±6

GPx1 mRNA

GPx1 mRNA

MUP mRNA

Nor

m

Nor

m

Nor

m

Ter

Ter

Ter

Gl mRNA(% of Norm)

100

7±10

023±

410

07±

1

DAPI MergeFLAG-PNRC1D HA-PNRC2

10 2210

09±

0 (% of Norm)