Fig 2: Smurf1 neddylated RRP9 to promote tumorigenesis.A, Smurf1 was deleted by lentivirus-coupled sgRNA. The expression of Smurf1 was detected by western blot to determine the efficiency of deletion. B–D, RNA was labeled with biotin and analyzed by fluorography in Smurf1 knockout cells. Bars represent means of radio labelled 18S rRNA ± S.D. from three experiments. E, average area of nucleolar organizing regions in indicated cells. Left panel, quantitative analysis of AgNOR indices. Right panel, images of silver staining. AgNOR from 20 cells was measured in each group. The scale bars represent 50 μm. F–H, CCK8 assay (F), colony formation assay (G), and cell migration assay (H) were performed in the indicated cells. Data are presented as means ± S.D. Results are from a representative experiment performed in triplicate. Image J was used to perform quantitative analysis. I, β-galactosidase (β-gal) assay was performed in the indicated cells. Image J was used to perform quantitative analysis. The differences between groups were assessed by one-way ANOVA test. p values were calculated by one-way ANOVA test (E, G, H, and I) and two-way ANOVA test (F). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. J, average area of nucleolar organizing regions in indicated cells. Up panel, quantitative analysis of AgNOR indices. Down panel, images of silver staining. AgNOR from 20 cells was measured in each group. The scale bars represent 50 μm. K–M, CCK8 assay (K), colony formation assay (L), and cell migration assay (M) were performed in the indicated cells. Data are presented as means ± S.D. Results are from a representative experiment performed in triplicate. Image J was used to perform quantitative analysis. p values were calculated by one-way ANOVA test (J, L, and M) and two-way ANOVA test (K). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig 3: Smurf1 interacts with RRP9.A and B, cellular extract from HCT116 cells was immunopurified with anti-Smurf1 antibody (ab117552) and then eluted. The eluates were resolved by SDS-PAGE and silver-stained. The protein bands were retrieved and analyzed by mass spectrometry. Immunopurified proteins were analyzed by western blotting using antibodies against the indicated proteins. C, immunoblot of anti-RRP9 immunoprecipitate from HCT116 cells. D, GST pull-down assays of His-Smurf1 with GST or GST-RRP9 are indicated. E, immunofluorescence of RRP9 and Smurf1 is indicated HCT116 cells. Scale bar, 25 μm. F–H, mapping the interaction binding regions between RRP9 and Smurf1. Shown is mapping the RRP9 binding region on Smurf1. Cell lysates from HCT116 cells transfected with Myc-tagged Smurf1 or Myc-tagged RRP9 deletion mutants were immunoprecipitated with anti-Myc followed by immunoblotting with anti-RRP9 or anti-Smurf1.

Fig 4: Smurf1 promotes RRP9 neddylation on K221.A, a schematic diagram of the lysine sites on RRP9 is shown. B, in vivo RRP9 neddylation assay. Immunoblot analysis of anti-Myc immunoprecipitate and WCL from HCT116 cells transfected with indicated constructs. C, RRP9 neddylation occurred on K221. Immunoblot analysis of anti-Myc immunoprecipitate and WCL from HCT116 transfected with indicated constructs. D, purified His-Smurf1 and GST-RRP9 WT or K221R proteins were incubated with Nedd8, Nedd8-E1/E2. Reactions were performed and analyzed by western blotting.

Fig 5: Smurf1 serves as an E3 ligase of RRP9 neddylation.A, HCT116 cells were treated with MG132 (0, 0.5, 1, 2 μM) for 8 h. Immunoblot analysis of whole cell lysates (WCL) from HCT116 cells. B, immunoblot of WCL from Smurf1-deleted HCT116 cells. C, immunoblot analysis of indicated proteins from the tissues of Smurf1-WT or Smurf1-KO mice. D, RRP9 neddylation was attenuated by MLN4924 (1 μM, 16 h). Immunoblot analysis of anti-RRP9 immunoprecipitate and WCL from HCT116 cells. E, immunoblot of anti-Myc immunoprecipitate and WCL from HCT116 cells transfected with indicated constructs. F, RRP9 neddylation was enhanced by the deletion of NEDP1. Immunoblot analysis of anti-Myc tagged RRP9 immunoprecipitate and WCL from HEK293T NEDP1+/+ and NEDP1−/− cells. G and H, RRP9 neddylation was reduced in Smurf1−/− (KO) or Smurf1C426A (KI) mice. Immunoblot analysis of anti-RRP9 immunoprecipitate and WCL from the tissues of Smurf1 WT, KO or KI mice. I, RRP9 neddylation was attenuated by deletion of UBA3, Ubc12, Smurf1. Immunoblot analysis of anti-RRP9 immunoprecipitate and WCL from HCT116 cells. J, overexpression Smurf1 increases RRP9 neddylation. Immunoblot of anti-Myc immunoprecipitate and WCL from HCT116 cells transfected with indicated constructs. K, in vitro covalent neddylation of RRP9. Purified His-Smurf1 and GST-RRP9 proteins were incubated with Nedd8, Nedd8-E1/E2. Reactions were performed as described in the Methods section. Samples were analyzed by western blotting with indicated antibody.