SMG-8 is required for SMG-1-mediated Upf1 phosphorylation in vivo. (A) HeLa TetOff cell lysates transfected with the siRNAs shown above the blots were analyzed by Western blotting with the antibody probes indicated. Anti-P-Upf1 antibody recognizes phosphorylated Ser1078 and Ser1096 residues of Upf1. (B) HeLa TetOff cells were treated with the siRNA indicated above the blot and subsequently transfected with plasmid expressing CBP-SMG-8r harboring a silent mutation in the siRNA targeting site. Cell lysates were analyzed by Western blotting with the antibodies indicated and the relative values of phospho-Upf1, normalized to Upf1 signals, were plotted. (A,B, right panels) Mean values ± SD from three independent experiments are shown.

Identification of SMG-8 and SMG-9, subunits of the SMG-1 complex (SMG1C). (A) HeLa cell lysates were immunoprecipitated with anti-SMG-1 antiserum (C or L) (Yamashita et al. 2001) or with preimmune serum (C preim. or L preim.). In vitro kinase reactions were then performed without substrates. (B) HeLa cell extracts were subjected to immunoprecipitation with anti-SMG-1-N antibody (Yamashita et al. 2001) or normal rabbit IgG (NRIgG). Coprecipitated proteins were detected by silver staining following SDS-PAGE. The signals indicated by arrows were analyzed by mass spectrometry. Asterisk indicates the SMG-1-N antibody background precipitation. (C) Schematic representation of SMG-8 and SMG-9. Gray boxes indicate conserved regions (CR) among metazoans. (D) HeLa TetOff cell extract was subjected to immunoprecipitation with three different anti-SMG-1 antisera (C, L, or N) or NRS. Immunoprecipitates were probed with the antibodies indicated. (E) HeLa TetOff cell extract was subjected to immunoprecipitation with anti-SMG-1-C antiserum or NRS. In vitro kinase reactions were performed with recovered immunocomplexes. After the kinase reactions, complexes were denatured in 2% SDS and then diluted 10-fold. Denatured proteins were subjected to the second immunoprecipitation with the antibodies indicated. Phosphorylated proteins were detected by autoradiography following SDS-PAGE. Schematic representation of this experiment is illustrated on the left. (F) HeLa TetOff cell lysates were immunoprecipitated with anti-SMG-9 antibody or NRIgG. Both immunoprecipitate (IP) and unbound fractions (Unb) were analyzed using the antibodies indicated.

SMG-8 is an inhibitor of SMG-1 kinase. (A) HeLa TetOff cells were transfected with the siRNAs indicated. SMG-1 complexes were immunoprecipitated with anti-SMG-1-C antiserum and analyzed by Western blotting. (NS) Nonsilencing siRNA; (pre-im) preimmune serum. (B) In vitro kinase assay with SMG-1 immunoprecipitates as the kinase source and GST-S1096 (GST fusion peptide containing Ser1096 of Upf1) as the substrate. (C) Quantification of the phosphorylation level of GST-S1096 in vitro. Relative values against NS-treated control are shown. Mean values ± SD from three independent experiments are shown.

SMG-8 and SMG-9 are components of the mRNA surveillance complex. (A,B) Schematic structure of the Upf1-C126S mutant (A, top panel). HeLa TetOff cells were transfected with plasmids encoding wild-type HA-Upf1 (WT) or HA-Upf1-C126S (CS). A minus sign (−) indicates empty vector. The cell extracts (input) were immunoprecipitated with anti-HA affinity matrix under the conditions of RNase-treatment with (B) or without (A) cycloheximide. Eluted immunocomplexes were probed with the antibodies shown on the left. (C,D) HeLa TetOff cells were transfected with siRNAs shown above the blots. The cell extracts (input) were immunoprecipitated (IP) with anti-Upf1 (5C3) antibody or with normal mouse IgG (NMIgG) in the presence of RNase and cycloheximide (C) or in the presence of cycloheximide, and the presence or absence of micrococcal nuclease (MN) (D). Input (the NS siRNA-treated cell extract equivalent to 10%, 3.3%, 1.1%, and 0.33% of the amount immunoprecipitated) and eluted immunoprecipitated fractions were analyzed by Western blotting with the antibodies indicated. (E) Model illustrating the ribosome:SURF complex on mRNPs during PTC recognition. Details are described in the text. (eRF) eRF1:eRF3 complex; (EJC) exon junction complex; (AUG) start codon; (Ter) termination codon; (PTC) premature termination codon.

SMG-8 knockdown causes the accumulation of the complex containing ribosome, eRF1, eRF3, Upf1, and EJC on mRNPs. (A–E) HeLa TetOff cells were transfected with the indicated siRNAs and the cell extracts were analyzed by Western blotting with the indicated antibody probes (left panel: input). The cell extracts were immunoprecipitated with anti-SMG-1-N antibody (A), anti-SMG-1-IP antibody (B), anti-Y14 (4C4) antibody (C), anti-HA antibody (12CA5, used as a control IgG), or anti-Upf1 (5C3) antibody (D,E) in the presence of RNase (A–D) or in the absence/presence of micrococcal nuclease (MN) (E). The cell extracts (left panel: input) and immunoprecipitates (right panel: IP) were analyzed by Western blotting with the antibodies shown on the left. (F) Schematic presentation of Upf1 complexes following SMG-8 siRNA treatment. SMG-8 knockdown leads to the accumulation of two distinct Upf1 complexes; SMG-1:SMG-9:Upf1 complex and ribosome:Upf1:eRF1:eRF3:EJC complex on mRNPs. Details are described in the text. (eRF) eRF1:eRF3 complex, (EJC) exon junction complex; (AUG) start codon; (Ter) termination codon, (PTC) premature termination codon.

SMG-8 and SMG-9 are required for NMD in mammalian cells. (A) Schematic structure of the β-globin gene (BGG) wild-type (WT)/PTC reporter plasmid used for the half-life assay. The ORF is represented by boxes and introns and UTRs are represented by lines. (B–D) HeLa TetOff cells were transfected with the siRNAs indicated and simultaneously transfected with Tet-inducible BGG PTC (B) or with BGG wild-type (C) reporter plasmid. After the addition of doxycycline to repress the transcription of reporter plasmids, total RNAs prepared at the times indicated were analyzed by Northern blotting using β-globin or GAPDH cDNA probes. The quantities of BGG mRNA, normalized to GADPH signals, were plotted (T1/2: half-life). (B,C) Mean values ± SD from three independent experiments are shown. (D) Forty-eight hours after siRNA transfection, cytoplasmic RNAs were prepared and analyzed by Northern blotting using GAS5 or GAPDH cDNA probes. (E,F) HeLa TetOff cells were transfected with the siRNAs together with plasmids expressing firefly luciferase based NMD reporter (E) or control reporter (F) and renilla luciferase plasmids and subjected to luciferase assay. Relative firefly luciferase activities, which were normalized by renilla luciferase activities, are shown graphically. Mean values ± SD from three independent experiments are shown. (G) HeLa TetOff cells transfected with the plasmids indicated above. Thirty-three hours after transfection, cells were lysed and analyzed by Western blotting with the indicated antibodies. Mean values ± SD from three independent experiments are shown.

SMG-8 is required for efficient NMD in C. elegans. (A) Alternative splicing of C. elegans rpl-12 pre-mRNAs. Rpl-12(PTC) mRNA contains a PTC and is a substrate of NMD. Rpl-12(+) does not contain a PTC and is not an NMD substrate. (B) Smg-8(RNAi) weakly inhibits C. elegans NMD. Unc-54(r293) smg-1(cc546ts) animals were fed bacteria expressing the indicated dsRNAs (lanes 2–4) or no dsRNA (lane 1). RNAs were prepared from these strains, converted to cDNA, and amplified with primers that flank the alternatively spliced rpl-12 intron. The positions of rpl-12(PTC) and rpl-12(+) PCR products following gel electrophoresis are indicated. Lane 5 is RT–PCR analysis of the deletion allele smg-2(r908) without RNAi treatment.

A model for the process from nonsense termination to mRNA decay. SMG1C, the SMG-1:SMG-8:SMG-9 complex, binds Upf1 before SURF formation. Then, the SMG1C:Upf1 complex interacts with eRF to form SURF, which associates with the ribosome and eEF2 on mRNPs containing the CBP20:CBP80 complex and PABPC1 after or during recognition of translation termination codons. SMG-8 is likely to mediate the recruitment of SMG-1 to the ribosome:SURF complex and to suppress SMG-1 kinase activity until the ribosome:SURF complex locates the EJC. SMG-9 is required for the efficient association between SMG-1 and SMG-8. The ribosome:SURF complex is most likely the translation termination complex operating during NMD, and looks for a downstream EJC to form the DECID (the ribosome:SURF:EJC complex). DECID formation likely induces SMG-1-dependent Upf1 phosphorylation, which leads to the recruitment of phospho-Upf1-specific binding proteins, such as SMG-5 and SMG-7 and the dissociation of the ribosome and eRF from Upf1 to advance mRNA decay. (AUG) Start codon; (Ter) termination codon; (PTC) premature termination codon; (EJC) exon junction complex; (eRF) eRF1:eRF3 complex; (S8) SMG-8; (S9) SMG-9; (P) phosphate group; (SURF) SMG-1:Upf1:eRF1:eRF3 complex; (DECID) decay-inducing complex.