Figures and data
Genetic screen identifies Uev1A as a crucial protector against RasG12V-induced nurse cell death.
(A) Schematic cartoon for Drosophila ovariole. During oogenesis, GSC undergoes asymmetric division to generate two daughter cells: one that self-renews and maintains GSC identity, and the other, called a cystoblast, that differentiates to support oogenesis. As differentiation progresses, each cystoblast performs four rounds of division with incomplete cytokinesis to produce 16 interconnected cystocytes, establishing a germline cyst. This germline cyst is then surrounded by epithelial follicle cells to form an egg chamber. Within each egg chamber, one of the 16 germ cells becomes the oocyte, while the remaining 15 differentiate into nurse cells. These nurse cells undergo G/S endocycling, becoming polyploid to aid in oocyte development. (B) Representative germarium and early-stage egg chamber with Flag-RasG12V overexpression driven by bam-GAL4-VP16. The red arrow denotes an early-stage egg chamber. (C) Genetic screening strategy. Genotype of “bam>RasG12V”: bam-GAL4-VP16/FM7;; UASp-RasG12V/TM6B. (D) Representative ovaries and egg chambers (DAPI staining). The red arrows in (D) denote degrading egg chambers. Scale bars: 200 μm. (E) Quantification data. 30 ovaries from 7-day-old flies were quantified for each genotype. Statistical significance was determined using t test (groups = 2) or one-way ANOVA (groups > 2): ** (P < 0.01) and *** (P < 0.001).
Uev1A protects against the nurse cell death induced by direct overexpression of RasG12V.
(A, C, and F) Representative samples (DAPI staining). (B) Molecular information of the uev1aΔ1 and uev1aΔ2 mutations. The red dashed lines represent nucleotide deletions. (D and G) Quantification data. 30 ovaries from 3-day-old flies were quantified for each genotype. Statistical significance was determined using t test (groups = 2) or one-way ANOVA (groups > 2): **** (P < 0.0001). (E) Protein sequence alignment of Uev1A, UBE2V1, and UBE2V2. It was performed using CLUSTALW and ESPript 3.0 software.
Roles of the DDR pathway and p53 in RasG12V-induced nurse cell death. (A) Representative ovaries (DAPI staining). Scale bars: 200 μm. (B) Quantification data. 30 ovaries from 3-day-old flies were quantified for each genotype. Statistical significance was determined using one-way ANOVA: **** (P < 0.0001). (C and E) Representative samples. Scale bars: 20 μm. (D) Schematic cartoon for uev1a-flag knock-in.
Uev1A collaborates with CycA to mitigate RasG12V-induced nurse cell death.
(A, C, E, and F) Representative ovaries. DAPI staining in (A and C). Scale bars: 200 μm in (A and C), 20 μm in (E and F). (B, D, and G) Quantification data. 30 ovaries (B and D) and 15 size-matched egg chambers (G) from 3-day-old flies were quantified for each genotype. Statistical significance was determined using t test (groups = 2) or one-way ANOVA (groups > 2): **** (P < 0.0001).
Uev1A collaborates with the APC/C complex to mitigate RasG12V-induced nurse cell death.
(A and C) Representative ovaries (DAPI staining). The red arrows in (A) denote degrading egg chambers. Scale bars: 200 μm. (B and D) Quantification data. 30 ovaries from 7-day-old (B) or 3-day-old (D) flies were quantified for each genotype. Statistical significance was determined using one-way ANOVA: **** (P < 0.0001).
Uev1A, Ben, and Cdc27 work together to degrade CycA through the proteasome.
(A and B) co-IP assays. The tagged proteins were co-expressed in S2 cells to assess physical interactions. As shown in (A), Uev1A interacts specifically with three APC/C subunits: Mr (APC2), Cdc16 (APC6), and Cdc23 (APC8). Assays in (B) demonstrate a physical interaction between CycA and Cdc27 (APC3). (C-E) CycA stability assays. CHX: a protein-synthesis inhibitor; MG132: a proteasome inhibitor; CQ: a lysosome inhibitor. In (D), the relative levels of CycA proteins were quantified using the following formula: (Mean gray value of the CycA/β-Actin band at n hours post-treatment) ÷ (Mean gray value of the CycA/β-Actin band at 0 hour). Three independent replicates were conducted at each timepoint, and statistical significance was determined using two-way ANOVA with multiple comparisons: **** (P < 0.0001). (F and G) CycA ubiquitination assays in S2 cells. As shown in (F), Cdc27 promotes CycA ubiquitination in a Uev1A/Ben-dependent manner. Assays in (G) indicate that the K11 and K63 of ubiquitin are required for CycA ubiquitination.
Uev1A inhibits the overgrowth of germline tumors induced by oncogenic RasG12V.
(A and C) Representative ovaries (DAPI staining). All images in (A) are of the same magnification. Scar bars in (C): 200 μm. (B) Quantification data for ovarian size. The largest 2D area of each ovary in a single confocal focal plane was scanned, and its size was measured using ImageJ. 30 ovaries from 3-day-old flies were analyzed for each genotype. (D) Representative samples. The GSCs within stem cell niches are outlined by yellow dashed lines. Both images are of the same magnification. (E) Quantification data for GSC numbers per germarium. Germ cells that directly contact cap cells and contain dot-like spectrosomes were counted as GSCs. 100 germaria from 14-day-old flies were quantified for each genotype. In (B and E), statistical significance was determined using t test: n.s. (P > 0.05) and * (P < 0.05). The online version of this article includes the following source data for figure 7: Source data 2. All genotypes. Source data 3. Raw quantification data.
Prognostic significance and tumor-suppressive effects of UBE2V1 and UBE2V2 on KRAS-mutant colorectal cancer.
(A) Kaplan-Meier analysis of relapsefree survival in KRAS-mutant colorectal cancer patients with high or low expression levels of UBE2V1 and UBE2V2. (B and E) The knock-down efficiency assays. The relative mRNA levels were normalized to GAPDH. (C-G) Assays to evaluate the effects of UBE2V1- and UBE2V2-RNAi on colony formation and cell viability in SW480 and HCT116 cells. In (B, C, E, and F), three independent replicates were conducted, and statistical significance was determined using t test. In (D and G), five (D) and six (G) independent replicates were conducted at each time point, and statistical significance was determined using two-way ANOVA with multiple comparisons. * (P < 0.05), *** (P < 0.001), and **** (P < 0.0001).
Overexpression of UBE2V1 or UBE2V2 suppresses the growth of KRAS-mutant colorectal cancer.
(A and B) Subcutaneous tumorigenesis assays in nude mice, where tumors were excised, photographed, and weighed 28 days after tumor cell injection. (C and D) Immunohistochemical staining to assess CycA expression and Ki-67 positivity in tumor tissues. All images in (C) are of the same magnification. In (B-1), six independent replicates were conducted, and statistical significance was determined using two-way ANOVA with multiple comparisons. In (B-2 and D), six independent replicates were conducted, and statistical significance was determined using one-way ANOVA. ** (P < 0.01), *** (P < 0.001), and **** (P < 0.0001). (E) Working model. By degrading CycA, Uev1A and the E3 APC/C complex counteract oncogenic Ras stimuli, thereby protecting against cell death in polyploid Drosophila nurse cells and suppressing overgrowth in diploid Drosophila germline and human colorectal tumor cells.
Uev1A protects against Yki3SA-induced nurse cell death.
(A) Representative ovaries (DAPI staining). The red arrows denote degrading egg chambers. Scale bars: 200 μm. (B) Quantification data. 30 ovaries from 7-day-old flies were quantified for each genotype. Statistical significance was determined using t test: * (P < 0.05) and **** (P < 0.0001). See also Source data 2 and 3.
Oncogenic RasG12V intrinsically triggers nurse cell death.
(A) Representative ovaries (DAPI staining). Scale bars: 200 μm. (B) Quantification data. 30 ovaries from 3-day-old flies were quantified for each genotype. Statistical significance was determined using t test: **** (P < 0.0001). See also Source data 2 and 3.
Uev1A is expressed in stretch follicle cells. Representative ovaries.
Uev1A does not directly degrade the RasG12V oncoproteins.
These experiments were performed at 29°C. All images are of the same magnification. See also Source data 2.
Expression pattern of Uev1A in germarium.
Representative sample. See also Source data 2.
Uev1A, Ben, and Cdc27 work together to protect nurse cells from death during normal oogenesis.
These experiments were performed at 29°C. (A) Representative ovaries (DAPI staining). The red arrows denote degrading egg chambers. Scale bars: 200 μm. (B) Quantification data. 30 ovaries from 7-day-old flies were quantified for each genotype. Statistical significance was determined using one-way ANOVA: **** (P < 0.0001). See also Source data 2 and 3.
Co-IP results.
The tagged proteins were co-expressed in S2 cells to assess physical interactions. Physical interaction was observed between Uev1A and Ben (A). No interaction was detected between Uev1A and Cdc27, Fzr, or Fzy (B, D, E), nor between Ben and Cdc27 (C).
RNAi efficiency assays.
The #1 dsRNAs were employed in RNAi assays targeting uev1a, ben, and cdc27 in Figure 6C, D, and F.
TCGA analysis comparing UBE2V1 and UBE2V2 expression levels in colorectal cancer patients with and without RAS mutations. The TCGA patient data were downloaded from the UCSC Xena website: the RNA-seq data (Version: 05-09-2024) and the somatic mutation data (Version: 08-05-2024). Statistical significance was determined using t test: n.s. (P > 0.05).
Knocking down either UBE2V1 or UBE2V2 alone mildly influences the growth of colorectal cancer cell lines.
(A and C) The knock-down efficiency assays. The relative mRNA levels were normalized to GAPDH. Three independent replicates were conducted, and statistical significance was determined using one-way ANOVA. (B and D) Assays to evaluate the effects of UBE2V1- and UBE2V2-RNAi on colony formation and cell viability in SW480 cells. Six independent replicates were conducted at each time point, and statistical significance was determined using two-way ANOVA with multiple comparisons. n.s. (P > 0.05), * (P < 0.05), ** (P < 0.01), *** (P < 0.001), and **** (P < 0.0001).
Validation of UBE2V1/2 overexpression in colorectal cancer cell lines.
(A) Western blotting to confirm the transient overexpression of UBE2V1 and UBE2V2 in SW480 and HCT116 cell lines. β-Actin was used as the loading control. (B) Western blotting to confirm the stable overexpression of UBE2V1 and UBE2V2 in SW480 cells, with UBE2V1-OE #1 and UBE2V2 #3 cell lines utilized in subcutaneous tumorigenesis assays. α-Tubulin was used as the loading control. In both (A) and (B), cells transfected with an empty overexpression vector served as the control.
UBE2V1/2 overexpression suppresses the growth of colorectal cancer cell lines.
(A and B) EdU incorporation assays to assess the effects of UBE2V1 and UBE2V2 overexpression (OE) on cell proliferation in SW480 and HCT116 cells. Empty OE vector was used as the control. All images in (A) are of the same magnification. (C-E) Assays to evaluate the effects of UBE2V1- and UBE2V2-OE on colony formation and cell viability in SW480 and HCT116 cells. In (B and D), three independent replicates were conducted, and statistical significance was determined using one-way ANOVA. In (E), five independent replicates were conducted, and statistical significance was determined using two-way ANOVA with multiple comparisons. * (P < 0.05), ** (P < 0.01), and **** (P < 0.0001).
