ATPase assay

The ATPase activity of UPF1 in the presence of its binding partners was determined by quantifying the amount of inorganic phosphate released upon ATP hydrolysis using a coupled colorimetric assay (EnzCheck Phosphate Kit, Thermo Fisher Scientific)²⁸. The protein mixtures (see below) were pre-incubated with 2 μg poly-U RNA, 40 nmol MESG (2-amino-6-mercapto-7-methylpurine ribonucleoside), and 0.5 U purine-nucleoside phosphorylase in reaction buffer (50 mM MES, pH 6.5, 50 mM potassium acetate, 5 mM magnesium acetate, 2 mM DTT) at 30 °C for 20 min. The reaction was initiated by the addition of ATP to a final concentration of 1 mM. Inorganic phosphate released from ATP hydrolysis reacted with MESG to produce 2-amino-6-mercapto-7-methylpurine, which was detected by measuring absorbance at 360 nm on an Infinite M1000 Pro (Tecan). The reaction was monitored over a 20 min period at 60-s intervals. The amount of UPF1 in every experiment was kept constant (6 pmol), and UPF2 was added in 1.25× excess of UPF1, wherever indicated. In Fig. 1, the full-length Stau1 protein was added in 10× excess of UPF1, while the experiments in Fig. 5 contained 7.5 pmol (1.25× of UPF1) of the dimeric Stau1 proteins. The end point of the ATPase reaction (20-min time-point) of the UPF1-UPF2 mixture was set to 100% and all other data points were normalized to this value. The raw data for all ATPase assays are provided as a Source Data file.