Phosphorylated UPF1-binding motifs trigger RNA decay in a UPF1-dependent manner. (A) The 3′ UTR of GADD45B mRNA contains four p-UPF1 binding motifs. (B,C) The decay of rabbit beta globin mRNA derived from pTET-BBB, pTET-BBB fused with GADD45B 3′ UTR, pTET-BBB fused with mutant GADD45B 3′ UTR deleted with indicated p-UPF1-binding motif, and pTET-BBB fused with mutant GADD45B 3′ UTR with substitution of contiguous adenylic acid sequence (A, stretch) were measured in the cells without siRNA transfection (B) or in the cells transfected with indicated siRNA (C). HeLa tet-off cells were transfected with a pGL4.13 luciferase (Luc) expression vector and indicated pTET-BBB rabbit beta globin reporter vectors. Doxycycline was then added to the medium to stop transcription from the tet-responsive promoter, and total RNA was collected at 0, 2, 4, and 6 h. qRT-PCR was performed to monitor Luc and beta globin levels and determine the relative RNA remaining. For each point, the beta globin mRNA level was normalized to the Luc RNA levels. Values represent mean ± SD from triplicate experiments. (D) RNA electrophoretic gel mobility shift assay was performed to determine the binding and release of UPF1 from the GADD45B 3′ UTR RNA (WT) or its adenine substitution mutant RNA (MUT). Five-tenths picomoles 32P-labeled RNAs were incubated with 500 ng UPF1 (lanes 3–8) or without UPF1 (lanes 1,2) for 15 min at 10°C. In lanes 5–8, 1 pmol nonlabeled WT GADD45B 3′ UTR RNA was added to the reactions after initial incubation and then incubated for another 30 min with ATP (lanes 7,8) or without ATP (lanes 5,6). Reaction solutions were then electrophoresed in a 4.5% native PAGE gel. The positions of the RNA probe and the UPF1-RNA complex are indicated on the left. The bar plot indicates the quantification of relative RNA release rate from UPF1 protein (adenine substitution mutant/WT 3′ UTR). Values represent the mean ± SD from triplicate experiments (biological replicates).
