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5a), depletion of SMG6 or XRN1 strongly GDC-0084 site enhanced complicated formation of UPF1 with SMG5 and SMG7 (evaluate lanes two, three with 1). UPF1 hyperphosphorylation value upon SMG5/7 depletion. If UPF1 hyperphosphorylation serves to boost the affinity of UPF1 for downstream components on stalls in the NMD pathway, then the capacity of UPF1 to undergo hyperphosphorylation must become increasingly significant for NMD because the availability of downstream elements is restricted. Certainly, as seen in the mRNA decay assays in Fig. 6a and Supplementary Fig. 6a, when low-level depletion of SMG7 or SMG5 didn't lower the efficiency of NMD within the presence of wild-type UPF1 (Fig. 6a, major left panel), several in the UPF1 [S/T]Q mutants became impaired in their NMD activity beneath these circumstances (quantified in Fig. 6b, examine white to grey bars) regardless of comparable SMG5/7 depletion efficiencies (Supplementary Fig. 6b). This impairment in NMD efficiency on SMG5 or SMG7 depletion became increasingly pronounced as groups of [S/T]Q to AQ mutations have been combined (compare person mutations in Fig.Dependent interactions (Supplementary Fig.Dependent interactions (Supplementary Fig. 5a), depletion of SMG6 or XRN1 strongly improved complicated formation of UPF1 with SMG5 and SMG7 (evaluate lanes 2, 3 with 1). Additionally, complex formation of UPF1 with SMG6 was enhanced on depletion of XRN1 (lane three) and, to a lesser extent, of SMG5/7 (lane 4). These observations show that manipulations that impair the NMD pathway downstream of UPF1 mRNA substrate binding result in increased RNA-independent association of UPF1 with downstream SMG5-7 aspects. To test whether the observed increase in association of UPF1 with downstream components is dependent on UPF1 phosphorylation, we compared the extent of SMG5-7 complex formation for UPF1 wild-type with two in the UPF1 [S/T]Q mutants: UPF1 [S/T]7,8,9,ten,11,17,18,19A (labelled UPF1-8ST4A in Fig. 5b), that is partially defective for NMD, and UPF1 [S/T] 1,2,7,8,9,ten,11,15,16,17,18,19A (UPF1-12ST4A), which is completely defective for NMD (Fig. 4). As seen in Fig. 5b, in contrast to wildtype UPF1 (lanes two, six and ten), the UPF1 [S/T]Q mutants fail to acquire enhanced association with SMG5 and SMG7 on depletion of SMG6 or XRN1 and rather preserve low amount of SMG5 and SMG7 association similar to that observed within the absence ofNATURE COMMUNICATIONS | DOI: ten.1038/ncommsSMG6 or XRN1 depletion (compare lanes 7, 8, 11, 12 with three, four). Similarly, as observed in Fig. 5c, wild-type and [S/T]Q mutant UPF1 can all be observed to associate with SMG6 (lanes 5-16), but only wild-type UPF1 shows enhanced association with SMG6 on depletion of XRN1 or SMG5/SMG7 (lanes six?). Hence, UPF1 seems to exhibit a basal degree of affinity for SMG5-7 proteins which is independent of hyperphosphorylation, consistent with recent observations for SMG6 (refs 32,49), which is further stimulated by phosphorylation. This was additional supported by in vitro pull-down assays, in which bacterially made UPF1 was observed to associate with SMG5/7 and SMG6 within a manner enhanced by phosphorylation with recombinant SQ-specific ATM kinase (Fig. 5d). Collectively, the observations in Fig.