Missense mutations impair UPF3B activity in NMD. a. Schematic representation of UPF3B protein structure with location of protein-protein interaction domains and amino acid changes. N-terminal N peptide and HA tag sequences are not shown. b. Schematic representation of the tethering assay mimicking NMD. λNUPF3B protein is tethered to Renilla luciferase mRNA with BoxB elements in the 3’ UTR expressed from phRL-TK-10BoxB (Test RNA) but is unable to bind mRNA expressed from phRL-TK lacking BoxB elements (Control RNA). Co-transfected pGL3-promoter or phrGFP serve as standards in dual luciferase and qPCR assays, respectively (Reference). c. Expression of λN-HA-UPF3B proteins. HeLa cells were transfected with pCI-λN-HA-UPF3B expression constructs and lysed after 48 h. Expression of UPF3B proteins and α-tubulin was analysed by 10 % SDS PAGE followed by Western blotting with anti-HA and anti-tubulin antibodies. d, e. Tethering assay: Luciferase activity. Hela cells were transfected with phRL-TK (d) or phRL-TK-10BoxB (e), together with reference plasmid pGL3-promoter and the pCI-λN-HA-UPF3B expression constructs. Luciferase activities were measured 48 h after transfection. Renilla luciferase activities were standardised with respect to firefly luciferase activity, and the activity in cells expressing UPF3B-Ala423 was defined as 1. Shown are average Renilla luciferase activities from three independent experiments. f, g: Tethering assay: Luciferase mRNA levels. HeLa cells were co-transfected with phRL-TK (f) or phRL-TK-10BoxB (g) but with phrGFP instead of pGL3-promoter. RNA was prepared 48 h later and mRNA levels were determined by qPCR. Luciferase mRNA levels were standardised with respect to GFP mRNA levels, and luciferase mRNA in cells expressing UPF3B-Ala423 was defined as 1. Shown are average luciferase mRNA levels from four independent experiments. Error bars indicate standard deviations, asterisks indicate values significantly different from luciferase activity or mRNA levels in the presence of UPF3B (one-way ANOVA followed by Dunnett's test; P < 0.05).