Fig. 5.

Loss of UPF1 processivity reduces NMD efficiency. a The levels of the expressed proteins, as tested with antibodies against the N-terminal TAP tag, were similar for UPF1 and UPF1^AKS→HPA, showing that the reduction in NMD efficiency is due to the mutation and not to a reduction in UPF1 expression levels. Two-fold dilutions of protein extracts were separated on 4–12% polyacrylamide gels, transferred and probed. The signal for an abundant protein, Zwf1, was used as loading control. b To test the ability of the UPF1^AKS→HPA variant to function in vivo in NMD, we used RT-QPCR to measure the steady-state levels of a natural NMD substrate, the transcript for the DAL7 gene. A upf1∆ strain was transformed with a control plasmid (pRS316), a plasmid expressing a region encompassing UPF1 helicase domain, and a plasmid expressing a mutant version (UPF1^AKS→HPA) and compared with a wild-type (WT) strain transformed with the control plasmid. The changes in DAL7 RNA levels compared with wild-type were measured in three independent experiments and used to calculate NMD efficiency, with average and standard deviation values shown as a bar plot