Phosphorylated UPF1-binding motifs trigger RNA decay in a UPF1-dependent manner. (A–E) HeLa tet-off cells were transfected with pTET-BBB vector or pTET-BBB+GADD45B 3′ UTR vector. pGL4.13 luciferase (Luc) expression vector was cotransfected as an internal control. Doxycycline was added to the medium to stop transcription from the tet-responsive promoter, and total RNA was collected at 0, 2, 4, and 6 h after addition of doxycycline. qRT-PCR was performed to monitor Luc and beta globin mRNA levels and determine the relative RNA remaining compared to time 0 h. For each point, the beta globin mRNA level was normalized to the Luc mRNA levels. The decay rates of rabbit beta globin mRNA derived from pTET-BBB or pTET-BBB+GADD45B 3′ UTR (GC-rich region) were measured in UPF1- (A), UPF2- (B), SMG1- (C), or STAU1- (D) depleted cells; or cycloheximide- (E) treated cells and compared with the control condition. Values represent the mean ± SD from triplicate experiments (biological replicates).
