Figure 7. Upf1 ATP hydrolysis is required for faster release of Upf1 from non-target over target mRNA.
(A) Northerns for β-globin and control reporter mRNAs in unbound fractions or coprecipitated with Flag-Upf1 WT from cell lysates treated with MgCl2/ATP for the number of minutes indicated above lanes. Graph under Northerns shows the ratio of βglobin mRNA recovery in IPs to the internal control β-GAP PTC mRNA after subtraction of background from negative control Flag IPs as a function of MgCl2/ATP-treatment duration, normalized to values at time 0. Data are represented as the normalized mean ratio +/− SEM for two to three biological replicates. P-value calculations were restricted to time points with triplicate measurements.
(B) Northerns of β-globin and control mRNAs in unbound fractions or coprecipitated with Flag-Upf1 from untreated lysates (-) or lysates treated with MgCl2/ATP or MgCl2/AMP PNP for 360 minutes. Graphs under Northerns represent mean ratios +/− SEM for triplicate biological repeats calculated as in panel A, normalized to values for untreated lysate samples.
(C) Similar to panel B comparing release of β-globin +/− HP mRNAs used in Figure 4B. Graphs under Northerns are from triplicate biological repeats normalized to values for AMP-PNP-treated samples.
Asterisks denote P-values: *≤0.1, **≤0.05, ***≤0.01 (paired student t-test, two-tailed). See also Figure S7
(D) Model depicting the ATPase-dependent mRNA discrimination step by Upf1 preceding NMD complex assembly. A second mRNA-selective commitment step might occur prior to mRNA degradation. See text for detail.