Fig. 6: TTK knockdown promotes ULK1 Exon 5 skipping leading to alternative splicing-nonsense-mediated mRNA decay (AS-NMD).

A Western blot analysis of ULK1 protein expression in RT-112 and UM-UC-3 cells stably transfected with shNC, shTTK#1, or shTTK#2. B qRT-PCR determination of ULK1 mRNA expression in RT-112 and UM-UC-3 cells stably transfected with shNC, shTTK#1, or shTTK#2. C Sashimi plot showing ULK1 exon 5 skipping in RT-112 and UM-UC-3 cells stably transfected with shNC (top) or shTTK (bottom). D Representative gel images from RT-PCR showing normal and abnormal splicing products of ULK1 mRNA in RT-112 and UM-UC-3 cells transfected with shNC, shTTK#1, or shTTK#2. Quantification of the percentage of RT-PCR products with exon 5 skipping among total ULK1 transcripts. E Schematic diagram illustrating how ULK1 exon 5 skipping may trigger NMD. Inclusion of exon 5 results in a functional protein with a termination codon in exon 28. Skipping exon 5 causes a frameshift, generating a premature termination codon at the start of exon 6, 12 nucleotides upstream of the last exon-exon junction. F, G RT-PCR detection of different isoforms of the indicated transcripts in RT-112 cells when NMD was inhibited. Cells were transfected with si-UPF1 or not after TTK knockdown. A number sign (#) indicates the NMD-sensitive isoform (F). Total levels of ULK1 transcripts in RT-112 cells after specific treatments were analyzed by qRT-PCR (G). H qRT-PCR measurement of ULK1 mRNA expression in RT-112 cells transfected with si-NC or si-UPF1 and treated with 10 μg/ml actinomycin D. Data are presented as mean ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.