Fig. 3
tiRNA-Gly directly binds to RBM17 and regulates RBM17 expression in PTC cells. A RNA pulldown assays are used to select proteins that interact with tiRNA-Gly in K1 cells. The red arrow marked the band where RBM17 located in tiRNA-Gly sense group in. Anti-sense and magnetic ball act as negative control. B The verification of RBM17 in RNA pulldown assays by western blot. 5% input acts as positive control. C RIP assays confirm the binding between endogenous RBM17 and tiRNA-Gly in K1 cells. Rabbit IgG acts as negative control, 10% input acts as positive control. Endogenous RBM17 antibody used in RIP assay. D RIP assays show deletion of the UHM (303–402 aa) of RBM17 significantly abolished the association between RBM17 full length (1–402 aa) and tiRNA-Gly. E Transfection tiRNA-Gly into K1 cells translocates RBM17 from cytoplasm into nucleus by immunofluorescence. Scale bar = 25 μm. F tiRNA-Gly is mainly located in cytoplasm by subcellular fractionation assay. β-actin served as the cytoplasmic internal control. U6 served as the nuclear internal control. Values are expressed as the mean ± SEM. G Transfection tiRNA-Gly into K1 cells decreases cytoplasmic RBM17 protein levels and increases nuclear RBM17 protein levels in K1 cells by western blot. GAPDH acts as cytoplasm control and Histone H3 as nucleus control. H and I Total RBM17 protein level is upregulated H) and total RBM17 mRNA level is not influenced I) after tiRNA-Gly is transfected into K1 cells. β-actin acts as the internal control. J K1 cells are treated with cycloheximide (CHX; 50 μg/ml) and transfected with tiRNA-Gly or scramble sequence (SCR) for indicated times. DMSO acts as a control group. Following treatment of CHX, the half-life of endogenous RBM17 protein is increased in tiRNA-Gly transfected K1 cells by Western blot. β-actin referred as a control. RBM17 qualification result shows in right. K K1 cells are treated with MG132 (20 μg/ml) and transfected with tiRNA-Gly or scramble sequence (SCR) for indicated times. DMSO acts as a control group. Following treatment with MG132, the accumulation of endogenous RBM17 in K1 cells transfected with tiRNA-Gly is largely higher compared to K1 cells transfected with scrambled siRNA by Western blot. β-actin referred as a control. RBM17 qualification result shows in right. L The binding of RBM17 and ubiquitinated protein is suppressed in K1 cells transfected with tiRNA-Gly by IP assay. Data are representative immunoblots of three independent assays. Values are triple replicated and displayed in mean ± SD. ***p < 0.001