The nonsense-mediated mRNA decay SMG-1 kinase is regulated by large-scale conformational changes controlled by SMG-8

Abstract

Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that regulates the degradation of mRNAs harboring premature translation termination codons. NMD also influences the expression of many physiological transcripts. SMG-1 is a large kinase essential to NMD that phosphorylates Upf1, which seems to be the definitive signal triggering mRNA decay. However, the regulation of the kinase activity of SMG-1 remains poorly understood. Here, we reveal the three-dimensional architecture of SMG-1 in complex with SMG-8 and SMG-9, and the structural mechanisms regulating SMG-1 kinase. A bent arm comprising a long region of HEAT (huntington, elongation factor 3, a subunit of PP2A and TOR1) repeats at the N terminus of SMG-1 functions as a scaffold for SMG-8 and SMG-9, and projects from the C-terminal core containing the phosphatidylinositol 3-kinase domain. SMG-9 seems to control the activity of SMG-1 indirectly through the recruitment of SMG-8 to the N-terminal HEAT repeat region of SMG-1. Notably, SMG-8 binding to the SMG-1:SMG-9 complex specifically down-regulates the kinase activity of SMG-1 on Upf1 without contacting the catalytic domain. Assembly of the SMG-1:SMG-8:SMG-9 complex induces a significant motion of the HEAT repeats that is signaled to the kinase domain. Thus, large-scale conformational changes induced by SMG-8 after SMG-9-mediated recruitment tune SMG-1 kinase activity to modulate NMD.

Figure 1.

Purification of SMG-1, SMG-1:SMG-9, and SMG1C. (A) SMG-1 primary structure and comparison with DNA-PKcs. In this study, the terms N-terminal and C-terminal are used to refer to the helical region and the conserved C-terminal domains, respectively. (B) Western blots of HeLa TetOff cell lysates immunodepleted with anti-SMG-1, anti-SMG-8, or anti-SMG-9 antibody, or NRIgG (normal rabbit immunoglobulin G). Immunodepleted lysates were analyzed using the antibodies indicated. Dilutions corresponding to 50%, 25%, 12.5%, or 6.25 of the NRIgG-depleted protein amount from the NRIgG-depleted lysate were loaded to assess the efficiency of the immunodepletion. (C–E) SDS gel after silver staining of the fractions eluted from the affinity column for the purification of the SMG1C complex (SMG-1:SMG-8:SMG-9) (C), the SMG-1:SMG-9 complex (D), or SMG-1 (E), all expressed in 293T cells.

Figure 2.

3D-EM structure of SMG-1:SMG-9. (A,B) Reference-free 2D averages of SMG-1:SMG-9 (A) compared with projections of the 3D structure (B) obtained after angular refinement.Bar, 10 nm. (C) Two views of the 3D structure of the SMG-1:SMG-9 complex obtained by angular refinement methods. (D) Reference-free 2D averages obtained from unstained specimens of the SMG-1:SMG-9 complex. Bar, 10 nm. (E) Cryo-EM structure of SMG-1:SMG-9.

Figure 3.

3D architecture of SMG-1 and the SMG-1:SMG-9 complex. (A) Immunolabeling of SMG-1:SMG-9 complexes using a monoclonal antibody targeting residues 3547–3657 at the C terminus of SMG-1. (B) Labeling of SMG-1:SMG-9 complexes using a 5-nm gold particle coupled to streptavidin binding the SBP tag at the N terminus of SMG-1. (C) Immunolabeling of SMG-1:SMG-9 complexes using a monoclonal antibody targeting the SBP tag at the N terminus of SMG-1. The bottom panels of A–C highlight the position of the protein and the antibody in the single images by removing the surrounding background. Antibodies targeting the C terminus are colored in green, whereas those labeling the N terminus are colored in blue. (D) Cartoon model of SMG-1:SMG-9 with the proposed location for the N-terminal and C-terminal domains of SMG-1 and SMG-9. (E) Structure of SMG-1 and its comparison with the structure of SMG-1:SMG-9. (*) The region of density in SMG-1:SMG-9 that is not present in SMG-1. (F) Difference map of SMG-1:SMG-9 and SMG-1 (purple) superimposed on the structure of SMG-1:SMG-9, shown as a transparency.

Figure 4.

3D architecture of the SMG1C complex. (A,B) Reference-free 2D averages of SMG1C (A) compared with projections of the 3D structure of SMG1C (B). Bar, 10 nm. (C) Cartoon of a 2D average of SMG1C. (D) Comparison and difference between 2D reference-free averages of SMG-1:SMG-9 (top left) and SMG-1:SMG-8:SMG-9 (top right). The difference image (orange) locates the position of SMG-8 in the SMG1C complex, and is represented superimposed on the average of SMG-1:SMG-9 (middle left) and SMG1C (middle right). Several 2D averages of SMG1C were cross-correlated with projections of the 3D structure of SMG1C, permitting the assignment of the mass detected by difference mapping, SMG-8, within the structure of SMG1C. All of the different averages used mapped the same density. (E) Views of the 3D structure of SMG1C compared with the compatible view in SMG-1:SMG-9. Color codes are as follows: The putative mapping of SMG-8 is colored in orange, the C-terminal head region is in green, the N-terminal arm domain is in blue, and SMG-9 is in purple.

Figure 5.

SMG-8 and SMG-9 bind the N-terminal region of SMG-1. (A) SBP-tagged and Flag-tagged SMG-1 plasmids used. (B–D) 293T cells were transfected with SBP-tagged (B) and Flag-tagged (C,D) SMG-1 plasmids shown in A, together with a plasmid expressing the siRNA targeted to the 3′ untranslated region (UTR) of SMG-1. Cells were lysed and pulled down with the streptavidin sepharose (B) or anti-Flag (C,D) antibody in the presence of RNaseA. Pulled-down products or cell lysates (input) were then probed with the antibodies shown on the right.

Figure 6.

Kinase activity of recombinant SMG-1:SMG-9 and SMG-1:SMG-8:SMG-9 complexes. (A) Fifty femtomoles of SMG-1 complexes was separated by SDS-PAGE and silver-stained. (B) In vitro kinase assay using 10 fmol of purified complexes and 30 pmol of GST-Upf1-S1096 peptide as the substrate. (C) Quantification of the phosphorylation level of GST-Upf1-S1096 in vitro. Relative values against the 5-min activity of the SMG-1:SMG-8:SMG-9 complex are shown. Mean values ± SD from three independent experiments are shown. (D) In vitro kinase assay using the indicated amount of purified SMG-8, 10 fmol of purified SMG-1:SMG-9 complex, and 30 pmol of GST-Upf1-S1096 peptide as a substrate for 10 min.

Figure 7.

Kinase activity of endogenous SMG-1:SMG-9 and SMG-1:SMG-8:SMG-9 complexes. (A) HeLa TetOff cells were transfected with the nonsilencing siRNA (for those experiments labeled as “no kinase” and “SMG-1:SMG-8:SMG-9 complex”) or siRNA targeted to SMG-8 (for the SMG-1:SMG-9 complex). SMG-1 complexes were immunoprecipitated with preimmune serum (labeled “no kinase,” since it should not immunoprecipitate SMG-1) or anti-SMG-1-C antiserum for the SMG-1:SMG-8:SMG-9 complex and the SMG-1:SMG-9 complex, and were analyzed by Western blotting. (B) In vitro kinase assay with SMG-1 immunoprecipitates using and SBP-Upf1 as a substrate. (C) Quantification of the phosphorylation level of SBP-Upf1 in vitro. Relative values against a nonsilencing siRNA-treated control are shown. Mean values from two independent experiments are shown.

Figure 8.

Model of the regulation of the kinase activity of SMG-1 through the assembly of the SMG1C complex. SMG-8 could be recruited to a preformed SMG-1:SMG-9 complex or as part of a pre-existing SMG-8:SMG-9 complex.