Diagrams show MDM2 (a1) and GNL3L single (b1) or multi-domain (c1) deletion mutants. Grey lines and numbers indicate the deleted regions and amino acid positions. Abbreviations: N, p53-binding; I1 and I2, intermediate-1 and -2; AZ, acidic-zinc finger; R, RING-finger; B, basic; C, coiled-coil; G, GTP-binding; and I, intermediate. CoIP showed that binding between GNL3L and MDM2 requires the I1, AZ, and I2 domains of MDM2 (a2) and the G- or I-domain of GNL3L (b2, c2, and c3).

(a) Confocal studies showed that GFP-fused GNL3L and FLAG-tagged MDM2 colocalize in the nucleoplasm of HCT116-8 cells and that coexpression of these two proteins does not change each other's distributions. Anti-fibrillarin staining labels the nucleolus in singly transfected cells. Bars show 5um. (b1) The exchange rate between the nucleolar and nucleoplasmic pools of MDM2 was measured in HCT116-8 cells by FLIP (fluorescence loss in photobleaching). The nucleolar signal of GFP-fused MDM2 was bleached repeatedly (circle) and the nucleoplasmic signal was recorded (rectangle). Time-sequenced images and their time intervals (in seconds) to the first bleaching pulse were shown. (b2) Knockdown of GNL3L (G3L-KD, in red) significantly increases the nucleolar-nucleoplasmic exchange rate of MDM2 (p < 0.01). (b3) Overexperssion of GNL3L exerts only a small effect on decreasing the exchange rate of MDM2 (p = 0.05). Error bars represent s.e.m. shown in one direction (arrows in graph). Y-axis represents the relative fluorescence index (RFI). Top arrows indicate bleaching pulses.

(a) The cell cycle phenotypes of control (siScr) and GNL3L (siG3L) knockdown were analyzed by propidium-iodide-labeled flow cytometry in HCT116-8 (p53^+/+) and HCT116-2 (p53^−/−) cells. GNL3L knockdown triggers G2/M arrest more in the p53^+/+ cells than in the p53^−/− cells. (b) Western blots confirm GNL3L knockdown efficiencies and reveal a slight decrease of MDM2 proteins in the p53-wild-type and null cells and an increase of phospho-Cdc2 (Y15) in the p53-wild-type cells by GNL3L knockdown. (c) The GNL3L knockdown effect on the p53 activity was determined by quantitative RT-PCR assays of several p53 downstream targets. GNL3L depletion up-regulates Bax, 14-3-3-σ, and p21 in a p53-dependent manner. *, p < 0.01; **, p < 0.001; ***, p < 0.0001.

Nine types of human cancers show a significant percentage of samples with increased GNL3L expression. Among them, the colorectal, gastric, and esophageal carcinomas also show a down-regulation of 14-3-3σ and p21. Bax expression is increased in most tumors. Y-axis represents the percentage of tumor samples whose expression of the target gene is 1.5-fold higher (grey bars) or lower (black bars) than that of normal tissues.

(a) MDM2 (Myc) and NS family proteins (HA) were co-immunoprecipitated (coIP) and immunoblotted (IB) by anti-tag antibodies in HEK293 cells. The results showed that MDM2 preferentially interacts with NS and GNL3L. (b) Endogenous binding between MDM2 and GNL3L was demonstrated by anti-MDM2 (left panel) and anti-GNL3L (right panel) coIP experiments. IgG and preimmune serum (pre-I) precipitated samples were used as controls. (c) Anti-FLAG coIP of GNL3L (HA), NS (Myc), and MDM2 (FLAG) showed that GNL3L and NS compete against each other for MDM2 binding. By comparison, GNL3L shows more binding with MDM2 than wild-type NS does (left panel) and less binding with MDM2 than NS-G256V does (right panel). NS-G256V is a nucleoplasm-mislocalized mutant of NS (Fig. S1b3). (d) Triple coIP of GNL3L (HA), MDM2 (FLAG), and p53 (Myc) by anti-HA (left panel) or anti-Myc antibody (right panel) shows that GNL3L may bind p53 indirectly through MDM2.

(a) Transient siRNA knockdown of GNL3L (siG3-2) in HeLa cells decreases the level of endogenous MDM2 proteins. This effect can be reversed by coexpression of a siG3-2-resistant GNL3L, G3L-siR. (b) Coexpression of GNL3L (HA) increases the protein level of endogenous MDM2 in a dose-dependent manner. (c) The effect of GNL3L on MDM2 protein stability was determined in H1299 cells transfected with MDM2 alone (Ctrl) or cotransfected with MDM2 and GNL3L (G3L). After cycloheximide treatment, the MDM2 protein amounts were measured, adjusted by their α-tubulin amounts, and expressed as percentages of the MDM2 protein amount at the 0h time-point (bottom). (d) The cycloheximide experiment showed that GNL3L knockdown in HCT116-8 cells significantly reduces the protein stability of endogenous MDM2 (n=3, p<0.005, Repeated Measures ANOVA) without affecting the stability of endogenous p53 (p=0.27). (e) HEK293 cells were transfected with the (His)₆-tagged ubiquitin, MDM2 and a control (shScr) or GNL3L-specific (shG3-a) shRNAmir construct. Ubiquitylated MDM2 products were precipitated by Ni²⁺ sepharose (His PD) and detected by anti-MDM2 antibody. GNL3L depletion increases the ubiquitylation of MDM2, and this effect can be rescued by an shRNA-resistant GNL3L (G3L-shR). (f) Overexpression of wild-type GNL3L decreases MDM2 polyubiquitylation compared to the control sample. (g) GNL3L knockdown (left panel) or overexpression (right panel) has no effect on the MDM2-induced p53 ubiquitylation.

(a) For in vivo ubiquitylation assays, HEK293 cells were transfected with the (His)₆- tagged ubiquitin, MDM2 (Myc), and GNL3L or NS-specific shRNA constructs. GNL3L knockdown shows a stronger effect in increasing MDM2 ubiquitylation than NS does. (b) The effects of GNL3L and NS overexpression on MDM2 ubiquitylation were compared using the same assay. Overexpression of wild-type and nucleoplasmic forms (G3L-dB and G3L-G253V) of GNL3L shows the same effect in decreasing the ubiquitylation of MDM2. On the other hand, nucleoplasmic forms of NS (NS-dB and NS-G256V) show much stronger effects compared not only to wild-type NS but also to wild-type and mutant GNL3L. (c) Endogenous coIP of MDM2 and GNL3L (or NS) showed that mitosis increases the coIP efficiency of NS and MDM2 more than it does on the coIP of GNL3L and MDM2.

(a1) qRT-PCR measurements revealed a compensatory up-regulation of GNL3L transcripts following NS knockdown in p53-wild-type and null HCT116 cells and U2OS cells but not in HeLa or HEK293 cells. Conversely, GNL3L knockdown shows no such effect on NS. (a2) The increase of GNL3L expression by NS knockdown was confirmed by western blots. (b) Mycophenolic acid (MPA) treatment triggers a reciprocal increase of GNL3L and decrease of NS in HCT116-8 and 2 cells. The MPA-induced GNL3L up-regulation can be reversed by MG132 treatment but not by NS overexpression. (c) GNL3L can functionally rescue the MDM2 ubiquitylation phenotype of NS-knockdown cells. (d) GNL3L and NS form a complex regulatory network on the MDM2-p53 pathway. Both proteins show similar activities in MDM2 stabilization but differ in their modes of regulation. Multiple points of feedback and cross-regulation exist. Arrows and X’s indicate excitatory or inhibitory functions, respectively, and the dashed line represents an event regulated by the nucleolar release mechanism.