Phospho-dependent and phospho-independent interactions of the helicase UPF1 with the NMD factors SMG5–SMG7 and SMG6

Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that recognizes mRNAs with premature stop codons and targets them for rapid degradation. Evidence from previous studies has converged on UPF1 as the central NMD factor. In human cells, the SMG1 kinase phosphorylates UPF1 at the N-terminal and C-terminal tails, in turn allowing the recruitment of the NMD factors SMG5, SMG6 and SMG7. To understand the molecular mechanisms, we recapitulated these steps of NMD in vitro using purified components. We find that a short C-terminal segment of phosphorylated UPF1 containing the last two Ser-Gln motifs is recognized by the heterodimer of SMG5 and SMG7 14–3–3-like proteins. In contrast, the SMG6 14–3–3-like domain is a monomer. The crystal structure indicates that the phosphoserine binding site of the SMG6 14–3–3-like domain is similar to that of SMG5 and can mediate a weak phospho-dependent interaction with UPF1. The dominant SMG6–UPF1 interaction is mediated by a low-complexity region bordering the 14–3–3-like domain of SMG6 and by the helicase domain and C-terminal tail of UPF1. This interaction is phosphorylation independent. Our study demonstrates that SMG5–SMG7 and SMG6 exhibit different and non-overlapping modes of UPF1 recognition, thus pointing at distinguished roles in integrating the complex NMD interaction network.

treatment with a combination of different proteases (trypsin, Asp-N, chymotrypsin and thermolysin). Orange and blue bars indicate the position of every threonine and serine residue, respectively. The black bars denote the identified phospho-sites, with half-length bars corresponding to sites that had identification and localization scores lower than the default 75% threshold. Mock phosphorylated UPF1 gave identical peptide coverage and no phospho-peptides were identified. The sequence of residues 1070-1118 of UPF1 and the positions of the SQ motifs therein are indicated.
(B) In vitro kinase assays (using γ-32 P ATP) of the UPF1 N-and C-terminal constructs (UPF1NT and UPF1CT) with purified SMG1 proteins (left panel). The corresponding Coomassie-stained gel of the radioactive kinase assay is shown in the right panel. The isolated ends of UPF1 are efficiently phosphorylated by SMG1 wt but not by SMG1 KD. The experiment was performed as in Figure 1C (described in the main text).
(C) MS/MS spectra for phosphorylated GST-UPF1CT4. The top panel denotes fragmentation of the peptide harboring the S1116 site and confirms the presence of a phospho-group on S1116. The bottom panel corresponds to the peptide encompassing the S1089 and S1096 sites and confirms the presence of a phospho-group on S1096.   in the TPR domain that are thought to bind the phospho-serine moiety (based on the structure of H.s. SMG7 TPR (1)), and black stars denote residues of SMG5 and SMG7 (only those mentioned in the text) that mediate interaction between the TPR domains of these two proteins (based on the structure of C.e. SMG5:7 TPR, (2)).
Conservation was determined based on the chemical property of amino acids.
Residues conserved among SMG6, SMG7 and SMG5 are shaded in dark orange while residues conserved only across SMG6 orthologues are shaded in light orange. (B) RNA-dependent ATPase assay of UPF1ΔN, in the absence and presence of UPF2(761-1227) and SMG6fl, performed using a coupled phosphate-detection assay (4). Reaction trends were determined using the 5-15 minute time points. The catalytic activity of UPF1 is enhanced upon addition of UPF2 but not upon addition of SMG6.

Figure S5
GST pull-down assays of GST-UPF1fl and SMG6fl in the presence of the EJC, UPF2 and two constructs of UPF3 (UPF3fl and UPF3RRM). The pull-down was performed as described in Figure 1C in the main text. The inputs and precipitates were analyzed ATPase assay 40 nmoles of MESG (2-amino-6-mercapto-7-methylpurine ribonucleoside) and 0.05U of purine-nucleoside phosphorylase (components of the EnzChek® Phosphate Assay kit, Life technologies) were pre-incubated with 2 µg poly-U RNA in 1X ATPase buffer (50 mM MES pH 6.5, 50 mM potassium acetate, 5 mM magnesium di-acetate, 2 mM DTT) at 30°C for 20 minutes, following which 10 pmoles of UPF1ΔN and 20 pmoles of UPF2(761-1227) or SMG6fl were added to the indicated samples. The reaction was initiated by the addition of 10 mM ATP. Generation of 2-amino-6mercapto-7-methylpurine from MESG and phosphate (released from ATP hydrolysis) was detected by measuring absorbance at 370 nm on a Genios Pro (Tecan). The reaction was allowed to proceed for 20 minutes and A 370 was measured at 60-second intervals.