Figures and data
Uev1A protects against the nurse cell death induced by oncogenic RasG12V.
(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, F, H, and K) Representative ovaries and egg chambers. The red arrows in (D) denote degrading egg chambers. Scale bars: 200 μm. (E, I, and L) Quantification data. 30 ovaries from 7-day-old (E) or 3-day-old (I and L) flies were quantified for each genotype. Statistical significance was determined using t test (groups = 2) or one-way ANOVA (groups > 2): ** (P < 0.01), *** (P < 0.001), and **** (P < 0.0001). (G) Molecular information of the uev1aΔ1 and uev1aΔ2 mutations. The red dashed lines represent nucleotide deletions. (J) Protein sequence alignment of Uev1A, UBE2V1, and UBE2V2. It was performed using CLUSTALW and ESPript 3.0 software. The online version of this article includes the following source data and figure supplement for figure 1:
Source data 1. Screen results.
Source data 2. All genotypes.
Source data 3. Raw quantification data.
Figure supplement 1. Uev1A protects against the nurse cell death induced by oncogenic Yki3SA.
Regulators that orchestrate the nurse cell death induced by oncogenic RasG12V.
(A, C, E, and G) Representative ovaries. The red arrows in (E) denote degrading egg chambers. Scale bars: 200 μm. (B, D, F, and H) Quantification data. 30 ovaries from 3-day-old (B, D, and H) or 7-day-old (F) flies were quantified for each genotype. Statistical significance was determined using t test (groups = 2) or one-way ANOVA (groups > 2): **** (P < 0.0001).
The online version of this article includes the following source data and figure supplements for figure 2:
Source data 2. All genotypes.
Source data 3. Raw quantification data.
Figure supplement 1. Oncogenic RasG12V intrinsically triggers nurse cell death.
Figure supplement 2. Roles of the DDR pathway and p53 in the nurse cell death induced by oncogenic RasG12V.
Figure supplement 3. Uev1A does not directly degrade the RasG12V oncoproteins.
Figure supplement 4. Uev1A, Ben, and the APC/C complex work together to protect nurse cells from death during normal oogenesis.
Uev1A, Ben, and Cdc27 work together to degrade CycA through the proteasome.
(A and B) co-IP results. (C) RNAi efficiency experiments. The #1 dsRNAs were employed in RNAi assays targeting uev1a, ben, and cdc27 in (D, E, and G). (D-F) CycA stability assays. In (E), 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). In (F), cells harvested six hours post-treatment were analyzed. (G and H) Ubiquitination assays for CycA in S2 cells.
The online version of this article includes the following source data and figure supplement for figure 3:
Source data 3. Raw quantification data.
Figure supplement 1. Co-IP results.
Uev1A inhibits the overgrowth of germline tumors induced by oncogenic RasG12V.
(A and C) Representative ovaries. 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 (NIH, Bethesda, MD, USA). 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). (F) Working model. The E2 Uev1A/Ben complex, ubiquitin, and the E3 APC/C complex work together to degrade CycA, thereby preventing apoptosis or senescence, while also inhibiting cell overgrowth induced by oncogenic Ras through the MAPK signaling.
The online version of this article includes the following source data for figure 4:
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 overall survival and relapse-free survival in KRAS-mutant colorectal cancer patients with high or low expression levels of UBE2V1 and UBE2V2. (B) 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). (C and D) 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 (C) are of the same magnification. (E-G) Assays to evaluate the effects of UBE2V1- and UBE2V2-OE on colony formation and cell viability in SW480 and HCT116 cells. (H and I) Subcutaneous tumorigenesis assays in nude mice, where tumors were excised, photographed, and weighed 28 days after tumor cell injection. (J and K) Immunohistochemical staining to assess CycA expression and Ki-67 positivity in tumor tissues. All images in (J) are of the same magnification. In (D, F, I-2, and K), statistical significance was determined using one-way ANOVA. In (G and I-1), statistical significance was determined using two-way ANOVA with multiple comparisons: * (P < 0.05), ** (P < 0.01), *** (P < 0.001), and **** (P < 0.0001).
The online version of this article includes the following source data and figure supplement for figure 5:
Source data 3. Raw quantification data.
Figure supplement 1. Validation of UBE2V1 and UBE2V2 overexpression in colorectal cancer cell lines.
Uev1A protects against the nurse cell death induced by oncogenic Yki3SA.
(A) Representative ovaries. 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. 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.
Roles of the DDR pathway and p53 in the nurse cell death induced by oncogenic RasG12V.
(A) Representative ovaries. 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) Schematic cartoon for uev1a-flag knock-in. (D and E) Representative samples. See also Source data 2 and 3.
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.
Uev1A, Ben, and the APC/C complex work together to protect nurse cells from death during normal oogenesis.
These experiments were performed at 29°C. (A) Representative ovaries. 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.
(A) Uev1A interacts with Ben. (B) Uev1A has no interaction with Cdc27. (C) Ben has no interaction with Cdc27. (D) Uev1A has no interaction with Fzr. (E) Uev1A has no interaction with Fzy.
Validation of UBE2V1 and UBE2V2 overexpression in colorectal cancer cell lines.
(A) Western blot analysis confirming the transient overexpression of UBE2V1 and UBE2V2 in SW480 and HCT116 colorectal cancer cell lines. β-Actin was used as the loading control. (B) Western blot analysis confirming 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.
