ATPase assay

6 pmol of the UPF1 proteins 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 (EnzChek Phosphate Assay kit, Invitrogen) in 1× ATPase 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. Generation of 2-amino-6-mercapto-7-methylpurine from MESG and phosphate (released from ATP hydrolysis) was detected by measuring absorbance at 360 nm on an Infinite M1000 Pro (Tecan). The reaction was allowed to proceed for 20 min and monitored during this period by measurement of A₃₆₀ at 60-s intervals. Wherever indicated, 7.5 pmol of UPF2 (1.25× of UPF1) were added to the reaction mixture. To determine the significance of the differences observed in ATPase activity, unpaired t-tests were performed and used to determine the two-tailed P values. The significance threshold was set at 0.05.