Communication with the Exon-Junction Complex and Activation of Nonsense-Mediated Decay by Human Upf Proteins Occur in the Cytoplasm  Guramrit Singh, Steffen Jakob, Mark G. Kleedehn, Jens Lykke-Andersen  Molecular Cell  Volume 27, Issue 5, Pages 780-792 (September 2007) DOI: 10.1016/j.molcel.2007.06.030 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 The EJC Interacts with the hUpf Complex through Multiple Contacts with hUpf3 (A) Western blots of HeLa cell extracts depleted with anti-hUpf or -EJC protein antibodies as indicated on the top of each lane (lanes 2–7; Pre-I, preimmune serum) and immunoblotted for the proteins indicated on the right. Lane 1, undepleted extract. (B) Western blots showing coimmunoprecipitation assays. Endogenous protein complexes were immunoprecipitated (IP) with the antibodies indicated on the left from HeLa cell extracts predepleted with the antibodies indicated above the panel (collectively labeled “Depletion”). The immunoprecipitates were probed for the proteins indicated on the right. Left lanes in each panel, 5% total extract (5% TE). (C) Anti-FLAG coimmunoprecipitation assays between FLAG-tagged full-length hUpf3b or hUpf3b321–470, as indicated on the top, and coexpressed Myc-tagged EJC proteins as indicated on the right from RNase A-treated HEK293T cell extracts. Cells expressing FLAG-MS2 were negative IP control (lanes 1 and 4). Immunoblotting was performed with anti-Myc antibody on pellet fractions (lanes 1–3) or 5% of the input extract (lanes 4–6). (D) A model depicting the interactions between the EJC and the hUpf complex. The dotted-line arrows depict indirect interactions (based on Figures 1B and 1C), while solid arrow indicates a direct interaction (see Figure 5). Molecular Cell 2007 27, 780-792DOI: (10.1016/j.molcel.2007.06.030) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Overexpression of hUpf Interaction Domains Specifically Inhibits Nonsense-Mediated mRNA Decay (A) Schematic representation of the hUpf-hUpf interaction domains (shown in light gray) that were used as dominant-negative polypeptides in this study. (B) Northern blots showing decay rates in HeLa Tet-off cells of β-globin mRNA with a PTC at codon 39 (β39) in the absence of any overexpressed polypeptide (none) or in the presence of coexpressed Myc-tagged hUpf fragments indicated on the left. Time points above the panels refer to the time after tetracycline-mediated transcriptional repression. β39 mRNA half-lives calculated after mRNA levels were normalized to the constitutively coexpressed βG mRNA are shown (in minutes) on the right of each panel. The fold increase in mRNA half-lives caused by the overexpressed polypeptides is averaged over at least three independent experiments and is indicated with standard deviation in parentheses on the right. (C) Northern blots showing the half-life of wild-type β-globin mRNA (βWT) using endogenous GAPDH mRNA as internal control. (D) Northern blots showing decay rates of β39 reporter mRNA in the presence of overexpressed polypeptides indicated on the left with mutations in key residues. (E) Northern blots showing decay rates of a β-globin reporter mRNA (βARE) with the AU-rich element from GM-CSF mRNA in the 3′UTR in the presence of hUpf fragments, GFP-TTP100–174, or no protein (none) as indicated on the left. Molecular Cell 2007 27, 780-792DOI: (10.1016/j.molcel.2007.06.030) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Overexpressed hUpf2-hUpf3b Interaction Domains Inhibit NMD in the Cytoplasm but Inhibit NMD Poorly when Localized in the Nucleus (A) Northern blots showing the decay rates of the β39 NMD substrate mRNA in the presence of DsRed-hUpf2756–1065 with or without an NLS as indicated on the left. DsRed fluorescence images (DsRed) on the left show the cellular distribution of the corresponding fusion proteins. Relative cytoplasmic concentration (% of total) of the DsRed-tagged protein quantified from at least 15 randomly chosen cells is given on each of the fluorescence images. The half-lives of β39 mRNA with fold increase in decay rate and standard deviation calculated as in Figure 2B are on the right. (B) Northern blots showing the decay rates of β39 mRNA in the presence of no exogenous protein (none) or 3×hUpf3b1–143 or NLS-tagged 3×hUpf3b1–143 proteins as indicated on the left and indirect immunofluorescence images using anti-Myc antibody showing the cellular localization with relative cytoplasmic concentrations of the polypeptides. (C) Northern blots showing decay rates of GPx1-46 mRNA in the presence of DsRed- or NLS-DsRed-tagged polypeptides as indicated on the left of each panel. Molecular Cell 2007 27, 780-792DOI: (10.1016/j.molcel.2007.06.030) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Nucleus-Retained hUpf1 Is Strongly Impaired in Supporting NMD (A) Northern blots showing the decay rates of β39 mRNA from cells expressing an siRNA against luciferase (top panel) or hUpf1 (bottom three panels) and the exogenous proteins indicated on the left. The half-lives and fold change in half-lives (with standard deviation) as compared to the luciferase siRNA experiment are shown on the right (three experiments). (B) Indirect immunofluorescence images showing the cellular distribution of the exogenously expressed Myc-tagged hUpf1 fusion proteins indicated on the bottom with their relative cytoplasmic concentrations (% of total, at least fifteen cells) indicated. (C) Western blots showing expression levels of endogenous and exogenous hUpf1 from cells expressing siRNAs and hUpf1 fusion proteins indicated on the top. Endogenous hUpf3b is a loading control. Molecular Cell 2007 27, 780-792DOI: (10.1016/j.molcel.2007.06.030) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 RNPS1 Directly Interacts via Its Serine-Rich Domain with hUpf3 Proteins (A) Autoradiograms of in vitro pull-down assays between recombinant proteins (GST, lanes 6–10; GST-hUpf3b, lanes 11–15) and in vitro-translated EJC proteins indicated on top of each lane. Lanes 1–5, 5% input. Lanes 6–15, pull-down fractions. The migration of the EJC proteins, hnRNP A1 (negative control), and GST-GFP (positive control) are indicated on the left. Asterisks denote truncated protein products from in vitro translation. The triangle on the right marks the pulled-down positive control GST-GFP, whereas arrows on the right side indicate pulled-down test proteins. (B) In vitro binding assays between recombinant GST (lanes 5–8) or GST-hUpf3b (lanes 9–12) and in vitro-translated RNPS1 fragments or full-length protein as indicated on the top of each lane. Lanes 1–4, 5% input. Lanes 5–12, pull-down fractions. A schematic of the domain structure of RNPS1 is shown on the top (Mayeda et al., 1999). The amino acid positions of the serine-rich domain (S domain) and the RNA recognition motif (RRM), which are shown as light gray rectangles, are given below. Molecular Cell 2007 27, 780-792DOI: (10.1016/j.molcel.2007.06.030) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Exogenously Expressed hUpf3b-EJC Interaction Domains Inhibit NMD in the Cytoplasm but Inhibit NMD Poorly when Concentrated in the Nucleus (A) A schematic illustrating the interactions between hUpf3b and EJC proteins. Dotted arrows indicate potentially indirect interactions. (B) Northern blots showing the decay rates of β39 mRNA in the presence of DsRed-fused hUpf3b-EJC interaction domains (with or without an NLS) as indicated on the left. DsRed fluorescence images with relative cytoplasmic concentrations are shown for each fusion protein. mRNA half-lives shown on the right are calculated as in Figure 2B. (C and D) Northern blots showing the decay rates of GPx1-46 (C) or βARE (D) mRNAs in the presence of DsRed-fused hUpf3b-EJC interaction domains indicated on the left. Molecular Cell 2007 27, 780-792DOI: (10.1016/j.molcel.2007.06.030) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 Communication with the EJC and Activation of NMD by the hUpf Complex Occur in the Cytoplasm Dominant-negative polypeptides that interfere with hUpf complex formation and its communication with the EJC inhibit NMD only when localized in the cytoplasm (indicated by inhibitory lines [ ┤]). A dotted line on the mRNA in the nucleus represents an intron. PTC, premature termination codon. Molecular Cell 2007 27, 780-792DOI: (10.1016/j.molcel.2007.06.030) Copyright © 2007 Elsevier Inc. Terms and Conditions