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SMG6 interacts with the exon junction complex via two conserved EJC-binding motifs (EBMs) required for nonsense-mediated mRNA decay - PubMed

  • ️Fri Jan 01 2010

. 2010 Nov 1;24(21):2440-50.

doi: 10.1101/gad.604610. Epub 2010 Oct 7.

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SMG6 interacts with the exon junction complex via two conserved EJC-binding motifs (EBMs) required for nonsense-mediated mRNA decay

Isao Kashima et al. Genes Dev. 2010.

Abstract

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that detects and degrades mRNAs containing premature stop codons (PTCs). In vertebrates, PTCs trigger efficient NMD when located upstream of an exon junction complex (EJC). Degradation of PTC-containing mRNAs requires the endonucleolytic activity of SMG6, a conserved NMD factor; nevertheless, the precise role for the EJC in NMD and how the SMG6 endonuclease is recruited to NMD targets have been unclear. Here we show that SMG6 interacts directly with the EJC via two conserved EJC-binding motifs (EBMs). We further show that the SMG6-EJC interaction is required for NMD. Our results reveal an unprecedented role for the EJC in recruiting the SMG6 endonuclease to NMD targets. More generally, our findings identify the EBM as a protein motif present in a handful of proteins, and suggest that EJCs establish multiple and mutually exclusive interactions with various protein partners, providing a plausible explanation for the myriad functions performed by this complex in post-transcriptional mRNA regulation.

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Figures

Figure 1.
Figure 1.

The SMG6 N-terminal domain is required for NMD. (A) Domain organization of SMG6. (N-term) N-terminal region; (14-3-3) domain with a 14-3-3-like fold; (Linker) linker region; (PIN) PIN domain. (B–D) HeLa cells were transfected with the indicated siRNAs plus a mixture of two plasmids: one expressing the NMD reporters (β-globin or TCR-β with or without PTC), and another expressing GFP-NXF1 as transfection control. Plasmids expressing siRNA-resistant, wild-type, or mutant versions of human SMG6 were included in the transfection mixtures, as indicated. These proteins are fused to a V5 tag. V5-MBP served as a control. The levels of wild-type or PTC-containing reporters were analyzed by Northern blot and normalized to those of GFP-NXF1 mRNA. The numbers in italics below B and D represent the normalized levels of the PTC-containing reporter relative to those of the wild-type, which were set to 100 for each condition. (C shows mean values ± standard deviation obtained in three independent experiments using the TCR-β reporter. Note that the normalized values of the wild-type reporter were set to 100 for each condition (white bar), but are shown only for control cells expressing MBP and treated with β-Gal siRNA. (E) The expression of wild-type or mutant V5-SMG6 was analyzed by Western blotting using an anti-V5 antibody. Cotransfected GFP-NXF1 served as a transfection control.

Figure 2.
Figure 2.

The N-terminal region of SMG6 confers binding to NMD factors and EJC components. (A–F) Interaction of V5-tagged SMG6 (wild type or deletion mutant) with HA-tagged NMD factors or endogenous Y14 and Magoh in human cells. Proteins were immunoprecipitated using anti-V5 antibodies. A V5-tagged MBP served as a negative control. The presence of endogenous Y14 and Magoh in the immunoprecipitates was analyzed by Western blotting using specific antibodies. In all panels, the protein bands observed at ∼55 kDa represent SMG6 degradation products comigrating with the immunoglobulin heavy chain that cross-react with the anti-V5 antibody.

Figure 3.
Figure 3.

EBMs in UPF3, SMG6, and additional proteins. Alignment of EBMs in UPF3 (A), SMG6 homologs (B), and other human proteins (C). The organisms are Homo sapiens (Hs), Gallus gallus (Gg), Danio rerio (Dr), Branchiostoma floridae (Bf), Ciona intestinalis (Ci), Strongylocentrotus purpuratus (Sp), Ixodes scapularis (Is), Apis mellifera (Am), Tribolium castaneum (Tc), Pediculus humanus (Ph), Caenorhabditis elegans (Ce), and Brugia malayi (Bm). Conserved residues of the motif are highlighted in blue. Positively charged residues flanking the motif are highlighted in cyan. Colored circles above the UPF3 sequences indicate the residues of human UPF3b that interact with Y14 (magenta), Magoh (green), and eIF4AIII (gray), according to Buchwald et al. (2010). Asterisks indicate residues mutated in this study.

Figure 4.
Figure 4.

SMG6 EBMs confer direct EJC binding. GST pull-downs of the preassembled EJC, with wild-type or mutant GST-SMG6 (amino acids 1–207). Inputs (20%) and bound fractions (100%) were analyzed on a 4%–12% gradient SDS-PAGE. GST-UPF3bΔN serves as a positive control for EJC binding. In lanes 1 and 5, the identical reaction, with EJC alone, shows the absence of nonspecific binding to the GST beads.

Figure 5.
Figure 5.

Mutations in the EBMs abolish EJC binding, but not the interaction with NMD factors. (A–G) Interaction of V5-tagged SMG6 (wild type or the indicated EBM mutants) with HA-tagged NMD factors or endogenous Y14 and Magoh in human cells. Proteins were immunoprecipitated using anti-V5 antibodies and analyzed as described in Figure 2.

Figure 6.
Figure 6.

Mutations in the EBMs strongly reduce the interaction of SMG6 with PABP. (A) V5-tagged SMG6 wild type or the indicated EBM mutants were expressed in human cells and immunoprecipitated using anti-V5 antibodies. The presence of endogenous PABP, Y14, and Magoh in the immunoprecipitates was analyzed by Western blotting. (B) UPF3b competes with SMG6 for binding to the EJC. GST pull-downs of the preassembled EJC with GST-UPF3bΔN or GST-SMG6 (1–207). (Lanes 1,2,5,6) To test for competition of EJC binding, the pull-downs with GST-UPF3bΔN were done in the absence or presence of untagged SMG6 (1–207). (Lanes 3,4,7,8) Conversely, the pull-downs with GST-SMG6 (1–207) were done in the absence or presence of MBP-UPF3bΔN. Samples were analyzed as described in Figure 4.

Figure 7.
Figure 7.

SMG6 interaction with the EJC is required for NMD. (A–D) Complementation assays were performed with wild-type SMG6 and various SMG6 mutants and the indicated NMD reporters, as described in Figure 1. An SMG6 protein carrying mutations in catalytic residues of the PIN domain and MBP served as controls. A and C show Northern blot analysis of representative RNA samples. Wild-type and PTC-containing mRNA levels were normalized to those of the GFP-NXF1 mRNA. For each condition, the normalized values of the wild-type reporter were set to 100 (white bar in B). Bshows mean values and standard deviation obtained in three independent experiments using the TCR-β reporter. The corresponding values for the β-globin reporter are shown in italics below C. (D) Western blotting showing the expression levels of recombinant proteins.

Figure 8.
Figure 8.

Interaction of GFP-tagged GIDRP88 and TDRD3 with endogenous Y14, Magoh, and PABP in human cells. (A,B) Proteins were immunoprecipitated using anti-GFP antibodies and analyzed as described in Figure 2. GFP-MBP and YFP-SMG6 served as negative and positive controls, respectively.

Figure 9.
Figure 9.

Model illustrating SMG6 interactions with NMD factors. NMD occurs on mRNAs containing PTCs. In vertebrates, PTCs trigger efficient NMD when located upstream of an EJC. The core EJC components are Y14, Magoh, BTZ, and eIF4AIII (not shown). In a prevailing model of NMD, UPF1 and SMG1 are recruited by ribosomes terminating translation prematurely through interactions with the release factors eRF1 and eRF3. In the presence of UPF2 and UPF3, presumably bound to downstream EJCs on the mRNA, SMG1 phosphorylates UPF1; this in turn recruits SMG5, SMG6, and SMG7. SMG5, SMG6, and SMG7 are thought to bind phosphorylated UPF1 through a common 14-3-3-like domain. SMG5 and SMG7 interact with each other, and may recruit components of the general mRNA decay machinery. In this study, we show that SMG6 is recruited to surveillance complexes through interactions mediated by its N-terminal domain. This domain contains two EBMs that mediate direct interactions with the EJC, and additional sequences that confer binding to the additional NMD factors. Moreover, SMG6 contains a C-terminal PIN domain, which exhibits endonuclease activity and cleaves the mRNA in the proximity of the PTC. Because SMG6 and UPF3 compete for binding to the EJC, UPF3 must be released from the EJC to allow SMG6 binding. The step of the pathway at which these interactions take place is not known.

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