Fig 1: SUMO1-modified Menin disrupts TR’s association with Dyskerin and GAR1A. A Venn diagram determined by mass spectrometry analysis displays differential proteins recruited to TR after Y603F-MEN1+SUMO1 or Y603D-MEN1+SUMO1 transfection in 293T cells.B. Pie charts show TR binding proteins in the Y603F-MEN1+SUMO1-transfected 293T sample, but not in the Y603D-MEN1+SUMO1-transfected 293Tsample.C. Western blotting of in vitro transcribed TR pulldown assay from whole cell lysates of HEK 293T cells with over-expressed mutant (Y603F, Y603D) Menin and SUMO1 shows decreased Dyskerin and GAR1 binding in Y603D-MEN1+SUMO1-transfected cells. Coilin binding shows no difference between Y603F vs Y603D. Xpress-tagged Menin shows increased binding in Y603D-Menin compared to Y603F-Menin although Menin expression shows no difference. β-actin was used as a loading control.D. Western blotting of in vitro transcribed TR pulldown assay from whole cell lysates of HEK 293T cells with over-expressed mutant (Y603D, K609R) Menin and SUMO1 shows increased Dyskerin and GAR1 binding in K609R-MEN1+SUMO1 transfected cells. Coilin binding shows no difference between Y603D vs K609R. Xpress-tagged Menin shows increased binding in Y603D-Menin compared to K609R-Menin although Menin expression shows no difference. β-actin was used as a loading control.E. Quantification of the Pearson correlation coefficient (r) of colocalization of Dyskerin and TR from Fig. 7F shows significantly less Dyskerin and TR binding in Y603D-MEN1+SUMO1 transfected 293T cells compared to mutant Y603F-MEN1+SUMO1 and K609R-MEN1+SUMO1 transfected cells.F. Representative IF-FISH images show significantly less Dyskerin and TR binding in Y603D-MEN1+SUMO1 transfected 293T cells compared to mutant Y603F-MEN1+SUMO1 and K609R-MEN1+SUMO1 transfected cells. The scale bar is 5 µm.Data information: In (C, D), molecular weight markers are shown at the right of each blot. In (E), colocalization was analyzed using ImageJ plugin ‘Coloc 2’. Data are presented as means ± SD from a one-way ANOVA followed by Tukey’s multiple comparison tests (** = p < 0.01, *** = p < 0.001). A probability of p < 0.05 was considered statistically significant.
Fig 2: Menin Phosphorylation and SUMO1 modification promote Menin association with telomerase RNA by increasing protein stabilityA. Phosphorylation and SUMO1 modification of Menin promote Menin-TR association. RNA pulldown assay using in vitro transcribed TR and overexpressed WT or mutant (Y603F, Y603D, K609R) Menin with or without SUMO1. Menin was analyzed with an Xpress antibody that binds the C-terminal Xpress Tag of the protein or a Menin antibody that binds the central region of Menin. The Western blot using Xpress antibody indicates that Y603D Menin with SUMO1 expression is greatly increased in TR pulldown complex compared to WT, Y603F and K609R Menin with or without SUMO1 expression. Y603D-Menin shows less expression in TR pulldown binding than Y603D-Menin+SUMO1. No significant changes were detected with Menin antibody. β-actin served as a loading control.B. Western blotting of cytoplasmic and nuclear protein extracts shows that phosphomimetic Y603D-MEN1 and SUMO1 promote Menin stability. HEK 293T cells were transfected with Xpress-tagged MEN1 (WT or Y603F, Y603D) with or without HA-tagged SUMO1. Lamin B served as a loading control.C. Cycloheximide chase assay showing significantly higher protein degradation in Y603F-Menin with or without SUMO1 compared to Y603D-Menin with or without SUMO1. Y603F and Y603D MEN1-transfected HEK 293T cells co-transfected with SUMO1 were treated with 50µg/mL cycloheximide for indicated time points. Β-actin served as a loading control.Data information: Molecular weight markers are shown at the right of each blot. The Y603D-Menin+SUMO1 lane in (A) was intentionally loaded with less sample in order to show similar intensities of Xpress-Menin in the Input.
Supplier Page from DNASU for MEN1 (Homo sapiens) in pDNR-Dual (Creator donor/master vector)