UPF1 Association with the Cap-Binding Protein, CBP80, Promotes Nonsense-Mediated mRNA Decay at Two Distinct Steps  Jungwook Hwang, Hanae Sato, Yalan Tang, Daiki Matsuda, Lynne E. Maquat  Molecular Cell  Volume 39, Issue 3, Pages 396-409 (August 2010) DOI: 10.1016/j.molcel.2010.07.004 Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 Mapping the Region of UPF1 that Binds CBP80 (A) HeLa cells (1 × 107/150 mm dish) that stably express FLAG-UPF1 were crosslinked (+) or not (–) using formaldehyde and subsequently lysed. Half of the lysate from noncrosslinked cells was treated with RNase A. (Upper) Treated and mock-treated lysates were subjected to western blotting (WB) before (–) or after immunoprecipitation (IP) using anti(α)-FLAG or, as a control for nonspecific IP, mouse (m)IgG. The five leftmost lanes analyzed 3-fold dilutions of lysate and demonstrate that western blotting conditions are semiquantitative. (Lower) RT-PCR analysis of RNA purified from lysates, where crosslinks were undone using heat prior to RNA purification. (B) Diagrams of full-length MYC-UPF1(1–1118) and four MYC-UPF1 deletion variants. Amino acids 115–244, which contain the CH domain, are denoted to bind UPF2 and eRF3. (C) Lysates of HeLa cells (1 × 107/150 mm dish) that had been transiently transfected with 5 μg of the specified pCMV-MYC-UPF1 plasmid were analyzed by western blotting using the specified antibody before or after IP using anti-CBP80 or, to control for nonspecific IP, normal rabbit serum (NRS). Dots (•) mark detectable MYC-UPF1 variants, and the asterisk (∗) specifies a nonspecific band that does not interfere with the analysis. (D) As in (B). (E) As in (C) but using 10 μg of different MYC-UPF1 deletion variants. Table S1 provides the relative amounts of cell lysate used before IP compared to after IP in each western blot. All results are representative of at least two independently performed experiments. Molecular Cell 2010 39, 396-409DOI: (10.1016/j.molcel.2010.07.004) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 MYC-UPF1(419–700) Inhibits the CoIP of UPF1 and CBP80, and Abrogates NMD (A) HeLa cells (1 × 107/150 mm dish) were transiently transfected with 10 μg of pCMV-MYC or pCMV-MYC-UPF1(419–700). Two days later, total-cell lysates were immunoprecipitated using anti(α)-CBP80 or, as a control for nonspecific IP, NRS. Western blotting (WB) was performed using the specified antibody. The five leftmost lanes analyzed decreasing amounts of protein before (–) IP. RT-PCR was used to quantitate the level of GAPDH mRNA. RNA was spiked with a small amount of E. coli RNA after IP, and the level of E. coli LACZ mRNA was used to control for variations in RNA recovery (Sato and Maquat, 2009). The level of GAPDH mRNA in each lysate was normalized to the level of LACZ mRNA, and the normalized level in lysates lacking MYC-UPF1(419–700) was defined as 100. The five leftmost lanes analyzed 2-fold dilutions of RNA. (B) As in (A). However, cells were also transfected with pCMV-MYC-UPF1(1–1118), lysates were immunoprecipitated using anti-MYC, and RT-PCR was not performed. (C) HeLa cells (2 × 106/100 mm dish) were transiently transfected with 5 μg of the specified pCMV-MYC plasmids, either 1 μg of pmCMV-Gl Norm and 1 μg of pmCMV-GPx1 Norm (where the TGA selenocysteine codon at position 46 was converted to TGC) or 1 μg of pmCMV-Gl Ter and 1 μg of pmCMV-GPx1 Ter (where the TGA at position 46 codon was converted to TAA), and 1 μg of phCMV-MUP. RT-PCR was used to normalize the level of Gl mRNA relative to MUP mRNA, where the normalized level of Gl Norm mRNA in the presence of each pCMV-MYC plasmid was defined as 100%. (D) Essentially the same as (C). However, each transfection included the specified amount of pFLAG-CBP80 and enough pFLAG to bring the total amount of transfecting DNA to 4 μg. Table S1 provides the relative amounts of cell lysate used before IP compared to after IP in each western blot. All results are representative of at least two independently performed experiments. Molecular Cell 2010 39, 396-409DOI: (10.1016/j.molcel.2010.07.004) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 Expressing MYC-UPF1(419–700) Decreases SMG1 and UPF1 Binding to eRF1 and eRF3 to Form SURF, and Decreases SMG1 and UPF1 Binding to an EJC (A) HEK293T cells (1 × 107/150 mm dish) were transiently transfected with 15 μg of pCMV-MYC or pCMV-MYC-UPF1(419–700) and 5 μg of pcDNA3.1-HA-SMG1. IPs were performed using anti(α)-HA or rat IgG in the presence of RNase A, and western blotting utilized the specified antibody before (–) or after IP. (B) As in (A), except that HA-SMG1 was not expressed, HeLa cells that stably express FLAG-UPF1 were used, and IPs were performed using anti-FLAG or mIgG. Notably, the arrow designating eRF1 points to the position of eRF1 in leftmost and rightmost lanes, which migrated more slowly than it did in the center lanes. (C) As in (A) except that IPs were performed using anti-Y14 or mIgG. Table S1 provides the relative amounts of cell lysate used before IP compared to after IP in each western blot. Results are representative of two independently performed experiments. Molecular Cell 2010 39, 396-409DOI: (10.1016/j.molcel.2010.07.004) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 FLAG-CBP80(664–790) Inhibits the CoIP of CBP80 and UPF1, and Abrogates NMD (A) Diagrams of full-length FLAG-CBP80(1–790), including the three domains (D1, D2, and D3) determined by structural predictions, and two deletion variants. Dots signify regions known to bind CBP20 (Mazza et al., 2001). (B) HeLa cells (1 × 107/150 mm dish) were transiently transfected with 10 μg of the specified pFLAG derivative and analyzed by western blotting before (–) or after IP using anti(α)-FLAG. (C) Essentially as in (B). However, lysates were subjected to IP using anti-UPF1 and, subsequently, western blotting using the specified antibody. (D) Essentially as (C) except using different antibodies. Additionally, GAPDH and E. coli LACZ mRNAs were analyzed using RT-PCR as in Figure 2A. (E) As in Figure 2C, except that FLAG-CBP80 derivatives were expressed instead of MYC-UPF1 derivatives. Table S1 provides the relative amounts of cell lysate used before IP compared to after IP in each western blot. Results are representative of two independently performed experiments. Molecular Cell 2010 39, 396-409DOI: (10.1016/j.molcel.2010.07.004) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Expressing FLAG-CBP80(664–790) Decreases SMG1 and UPF1 Binding to eRF1 and eRF3 to Form SURF, and Decreases SMG1 and UPF1 Binding to an EJC (A) HEK293T cells (1 × 107/150 mm dish) were transiently transfected with 15 μg of pFLAG or pFLAG-CBP80(664–790) and 5 μg of pcDNA3.1-HA-SMG1. IPs were performed as in Figure 3A. (B) As in (A), except that HeLa cells were cotransfected with 5 μg of MYC-UPF1(1–1118), HA-SMG1 was not expressed, and IPs were performed using anti-MYC or mIgG. (C) As in (B), except that IPs were performed using anti-Y14 or mIgG. Table S1 provides the relative amounts of cell lysate used before IP compared to after IP in each western blot. Results are representative of three independently performed experiments. Molecular Cell 2010 39, 396-409DOI: (10.1016/j.molcel.2010.07.004) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 UPF1 Binds Gl Ter mRNA More Efficiently Than It Binds Gl Norm mRNA, and Inhibiting the Interaction of UPF1 with CBP80 Inhibits UPF1 Binding to NMD Targets (A) HeLa cells (1 × 107/150 mm dish) were transiently transfected with (1) 2 μg of pmCMV-Gl Norm and 2 μg of pmCMV-GPx1 Norm or 2 μg of pmCMV-Gl Ter and 2 μg of pmCMV-GPx1 Ter and (2) 2 μg of phCMV-MUP. Nuclear lysates (Gl mRNAs) or total-cell lysates (GPx1 mRNAs) were subjected to IP, or not (–), using anti(α)-UPF1 or, to control for nonspecific IP, NRS. Western blotting (WB) was performed using the specified antibodies, and RT-PCR was used to quantitate Gl, GPx1, and MUP mRNAs. (B) As in (A), except that nuclear lysates were analyzed from transfections that included 15 μg of pCMV-MYC or pCMV-MYC-UPF1(419–700) and 2 μg of pmCMV-Gl Ter and 2 μg of phCMV-MUP. IPs were performed using anti-UPF1 or NRS, and the levels of GAPDH and LACZ mRNAs were analyzed as in Figure 4D. (C) Diagram of Gl mRNA, where boxes denote exons, and the upward-pointing arrow specifies the position of oligo-directed RNase H cleavage within exon 1. Open arrowheads represent primers used to generate the RT-PCR product of full-length Gl mRNA (636 bp), its 5′ cleavage product (135 bp) or its 3′ cleavage product (501 bp). (D) As in (B). However, protein and RNA were analyzed without (−) or with (+) oligo-directed RNase H cleavage and without (−) or with subsequent IP using anti-UPF1 or, to control for nonspecific IP, NRS. Results are representative of at least two independently performed experiments. Molecular Cell 2010 39, 396-409DOI: (10.1016/j.molcel.2010.07.004) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 7 Model for CBP80-Promoted mRNP Rearrangements during NMD During the pioneer round of translation, ribosomes engage mRNA that is bound by the cap-binding heterodimer CBP80-CBP20 and, should the mRNA have been generated by pre-mRNA splicing, at least one EJC. By the time ribosomes reach a PTC, CBP80 of CBC interacts directly but transiently or weakly with the NMD factor UPF1 (which may already be associated with its kinase, SMG1). The CBP80-UPF1 interaction promotes the binding of UPF1 and SMG1 to the eRF1-eRF3 heterodimer that is poised at the PTC so as to form the SURF complex. It is possible, but not certain, that the same UPF1 molecule that binds CBP80 also forms SURF. The joining of SMG1-UPF1 to eRF1-eRF3 appears to be in competition with the joining of poly(A) tail-bound PABPC1 to eRF1-eRF3, the latter of which likely occurs predominantly at termination codons that do not trigger NMD (Ivanov et al., 2008; Singh et al., 2008). CBP80 subsequently promotes the joining of SMG1-UPF1 to a PTC-distal EJC, which generally consists of the NMD factors UPF2 and either UPF3 or UPF3X, and an interaction of SMG1 and UPF1 directly with UPF2 is shown. In agreement with Kashima et al. (2006), we found no convincing evidence that eRF1 or eRF3 accompanys SMG1-UPF1 to the EJC. However, we did detect an association of CBP80 with the EJC, as we did with SURF. Therefore, CBP80 appears to chaperone SMG1-UPF1 first to eRF1-eRF3 to form SURF and subsequently to a PTC-distal EJC. SMG1-UPF1 binding to the EJC triggers UPF1 phosphorylation (yellow-encircled “P”). UPF1 phosphorylation inhibits further translation initiation events at the mRNA 5′ end and enhances the efficiency of mRNA decay (Isken et al., 2008). AUG, translation initiation codon; Norm Ter, normal termination codon; PABPN1 is likely bound to the poly(A) tail along with PABPC1 (Hosoda et al., 2006; Sato and Maquat, 2009) but is not shown. Molecular Cell 2010 39, 396-409DOI: (10.1016/j.molcel.2010.07.004) Copyright © 2010 Elsevier Inc. Terms and Conditions