Fig. 5
From: Insights into the assembly and architecture of a Staufen-mediated mRNA decay (SMD)-competent mRNP
A pivotal role for UPF2 in the SMD pathway. a GST-pulldown assays of Stau1 with GST-UPF1CH and GST-UPF2s in the absence and presence of dsRNA. The dsRNA was partially labeled with 32P to enable its detection by autoradiography (lower panels of input and precipitate). As before, proteins were detected by staining with Coomassie Brilliant Blue (CBB). The co-precipitation of dsRNA with UPF1 is significantly enhanced in the presence of UPF2 due to strong interactions between UPF2 and Stau1 in the presence of dsRNA. b RNA-dependent ATPase of UPF1 in complex with UPF2 and Stau1, performed using an enzyme-coupled phosphate detection assay. The ATPase activity of UPF1 in the presence of either UPF2 or Stau1 served as controls. The data points and their error bars represent the mean values and standard deviation (s.d.) from three independent experiments. c Quantitative (q) PCRs to determine levels of known ADAR1/Stau1 targets, XIAP, MDM2, CCNG1, and RAD51, upon knockdown of SMD proteins in U2OS cells. Target mRNA levels were normalized to that of the GAPDH transcript in every case. The control siRNA refers to a scrambled sequence that does not specifically target any transcript. Knockdown of UPF2 in combination with ADAR1 leads to increase in the levels of SMD target. The ADAR1/Stau1 knockdown was performed as a positive control. The data were obtained from duplicates of the indicated number of biological replicates, with error bars denoting the standard deviation (s.d.) between the biological replicates. Individual data points are represented as solid circles, while the mean of each data series is represented as a column. The differences in mRNA levels between the ADAR1 knockdown and the ADAR1/UPF2 or the ADAR1/Stau1 knockdown samples is significant, as indicated by the p values obtained from unpaired t tests. The source data for a, b are provided as a source data file
