Fig. 1. Generation of an NMD reporter for Drosophila cells. (A) Schematic representation of the adh gene and the reporter used in this study. Black boxes, exons; white boxes, adh 5′- or 3′-UTRs; gray boxes, sequences derived from vector pAc5.1b; IN, introns. The PTC-containing constructs correspond to adh-nBR114 and adh-n4 (Brogna, 1999). (B) S2 cells constitutively expressing adh-wt or the PTC reporters were incubated with actinomycin D (5 µg/ml) for the times indicated above the lanes. Total RNA samples were isolated and analyzed by northern blot using probes specific for adh and rp49 mRNAs. (C) The levels of adh-wt, adh-n4 and adh-nBR114 were quantitated, normalized to the levels of rp49 mRNA (whose levels do not change relative to 18S rRNA within the time frame of the experiment) and plotted as a function of time. The half-lives of the mRNAs are indicated. (D) S2 cells constitutively expressing adh-wt or the PTC reporters were incubated with cycloheximide (CHX, 100 µg/ml) for 45 min. Total RNA samples were analyzed by northern blot. The numbers below the lanes indicate the levels of adh transcripts normalized to those of rp49 mRNA. These values were set to 100% in untreated cells.

Fig. 2. Adh reporters are subjected to NMD in Drosophila cells. (A–C) S2 cell lines expressing adh-wt, adh-n4 or adh-nBR114 were transfected with dsRNAs corresponding to UPF1 or UPF2 as indicated. Transfections were carried out in duplicate. Total RNA samples were analyzed by northern blot as described in Figure 1.

Fig. 3. Phylogenetic tree and domain organization of the SMG5–7 proteins. (A) The two identified orthologous groups are shown in red. Only one and a half TPR repeats can be identified automatically and are indicated in dark blue within the conserved helical N-terminal domain (cyan). The C.elegans sequences are underlined and are known to be involved in NMD (Cali et al., 1999; Page et al., 1999; Anders et al., 2003). Human and Drosophila sequences characterized in this study are shown in gray boxes. The vertical black bars on the schematic of the protein domains indicate that part of the protein sequence is not drawn to scale. The number of amino acid residues is shown on the right. Scale bar: 200 amino acids. TPR, tetratricopeptide repeats; PIN, PilT-N-terminus; Ce, Caenorhabditis elegans; Ci, Ciona intestinalis; Dm, Drosophila melanogaster; Fr, Fugu rubripes; Hs, Homo sapiens. (B) Multiple sequence alignment of the TPR-like region. The accession number of each sequence is indicated, as well as the species and the start and end points of the sequences. For the sequences of C.intestinalis and F.rubripes, no accession numbers are shown, as the predictions were performed directly on the genomic sequences using GeneWise (Birney et al., 1996). The consensus in 70% of the sequences is shown below the multialignment; h, l, p, +, – represent hydrophobic, aliphatic, polar, positive and negative residues, respectively. Aliphatic residues are highlighted in cyan, hydrophobic in blue, polar in green, negative in pink, and positive in red. The conserved glycines are highlighted in orange. The secondary structure prediction (Sec.Str.Pred.) is taken from the consensus of the multiple alignment (H, helices predicted with an accuracy >82%; h, helices predicted with an accuracy ≤82%]. The blue bars above the sequences show the position of the one and a half TPR repeats identified by Clissold and Ponting (2000).

Fig. 4. SMG1, SMG5 and SMG6 are essential for NMD in Drosophila cells. (A) S2 cell lines expressing adh-wt or adh-n4 were transfected with the dsRNAs indicated above the lanes. RNA samples were analyzed by northern blot, as described in Figure 2. (B) The steady-state levels of adh-wt and adh-n4 mRNAs were quantitated in at least three independent experiments and normalized to those of rp49 mRNA. For each experiment, the normalized values (norm.) obtained for n4 mRNA were divided by those obtained for adh-wt, to correct for potential non-specific effects of the depletions. This ratio was set arbitrarily to a value of 1 in control cells. The mean values ± SDs are shown. (C) S2 cell lines expressing adh-wt or adh-n4 were transfected with dsRNAs specific for UPF1 and SMG6. Seven days after addition of dsRNAs, treated and untreated cells were incubated with actinomycin D (5 µg/ml) for the times indicated above the lanes. Total RNA samples were isolated and analyzed by northern blot as described in Figure 1D.

Fig. 5. Human SMG5–7 are required for NMD in HeLa cells. (A) HeLa cells were transfected with the indicated siRNAs and plasmids expressing either β-globin-wt or β-globin-NS39 mRNAs. A plasmid expressing GFP–NXF1 was co-transfected to correct for differences in transfection efficiencies. RNA samples were analyzed by northern blot using β-globin and GFP probes. The numbers below the lanes indicate the levels of β-globin-wt or NS39 transcripts normalized to those of GFP–NXF1 mRNA and set to 100% in cells treated with an unrelated siRNA (–). (B) The steady-state levels of β-globin-wt or NS39 mRNAs were quantitated in at least three independent experiments and normalized to those of the GFP–NXF1 mRNA. The normalized values (norm.) obtained for NS39 were divided by those obtained for the wild-type mRNA. The mean values ± SDs are shown.

Fig. 6. The individual components of the EJC are dispensable for NMD in Drosophila. (A and B) S2 cells expressing adh-wt or adh-n4 were transfected with dsRNA specific for Y14, RNPS1, REF1, SRm160 or DEK, or with a mixture of dsRNAs (lanes 9 and 10) as indicated. Protein samples from total lysates of untreated or depleted cells (adh-n4 cell line) were analyzed by western blot using antibodies described in Gatfield and Izaurralde (2002). The anti-SRm160 antibody cross-reacts with three bands that are all depleted in cells treated with SRm160 dsRNA, suggesting that these bands may represent modified and/or alternatively spliced forms of the protein. Tubulin served as a loading control. In lanes 1–3, dilutions of the samples isolated on day 0 were loaded to estimate the efficiency of the depletion. (C and D) RNA samples isolated from depleted cells shown in (A) and (B) were isolated and analyzed by northern blot as described in Figure 2. Samples isolated from cells treated with UPF1 or UPF2 dsRNAs were analyzed in parallel. The numbers below the lanes indicate the levels of adh transcripts normalized to those of rp49 mRNA. (E) Normalized values obtained in three independent experiments for n4 were divided by those obtained for adh-wt. These ratios were set arbitrarily to 1 in control cells. (F) S2 cells expressing adh-n4 were transfected with dsRNA specific for UPF1, or with mixtures of dsRNAs as indicated. RNA samples were analyzed by northern blot. The numbers below the lanes indicate the levels of adh-n4 transcript normalized to those of rp49 mRNA.

Fig. 7. PTCs are defined independently of downstream exon boundaries in Drosophila cells. (A) Schematic representation of the reporters lacking a downstream intron. Symbols are as in Figure 1A. (B) S2 cells expressing adh-wt, adh-n4 or adh-nBR114 reporters lacking intron 3 (adhΔIN3-wt, adhΔIN3-n4, adhΔIN3-nBR114) were treated with UPF1, UPF2 or UPF3 dsRNAs. Total RNA samples were analyzed as described in Figure 2. (C) The steady-state levels of adhΔIN3-n4 and adhΔIN3-nBR114 mRNAs were quantitated in at least three independent experiments and normalized to those of rp49 mRNA. These normalized values (adhΔIN3-PTC norm.) were divided by those obtained for adhΔIN3-wt. The mean values ± SDs are shown. (D–F) S2 cells constitutively expressing adhΔIN3-wt, adhΔIN3-n4 or adhΔIN3-nBR114 were treated with UPF1 dsRNA. Seven days after addition of UPF1 dsRNA, treated or untreated cells (control) were incubated with actinomycin D (5 µg/ml) for the times indicated above the lanes. Total RNA samples were isolated and analyzed by northern blot.

Fig. 8. PTCs can be recognized on a heterologous mRNA. (A) S2 cells expressing CAT-wt or CAT-72 mRNAs were treated with UPF1, UPF2 or UPF3 dsRNAs. Total RNA samples were analyzed by northern blot using probes specific for CAT and rp49 mRNAs. The numbers below the lanes indicate the levels of the CAT transcripts normalized to those of rp49 mRNA. (B) S2 cells expressing GFP-wt or GFP-52 mRNAs were treated with UPF1, UPF2 or UPF3 dsRNAs. The levels of the GFP transcripts normalized to those of rp49 mRNA are indicated below the lanes. (C) The levels of GFP-wt and GFP-52 mRNAs were quantitated in at least three independent experiments and normalized to those of rp49 mRNA. These ratios were set arbitrarily to 1 in control cells. The mean values ± SDs are shown. (D) S2 cell lines expressing GFP-wt or GFP-52 were treated with the dsRNAs indicated above the lanes. Total RNA samples were analyzed as described in (B).