EMBO/SourceData huggingface dataset:
Figure 9(A) Western blot results show knockdown of ATM attenuated 100 μM H2O2 induced T2609 and T2647 phosphorylation of DNA-PKcs in HeLa cells.(C,D) PLA Analysis shows that the interaction between DNA-PKcs and ANT2 in MEF cells was temporarily disrupted by treatment of 10Gy IR. DNA-PKcs3A/3A remained interaction with ANT2 after IR. The bar graphs were generated from three independent biological replicates analyses; data are represented as mean ± SD. Statistical significance was established using a two-way analysis of variance (ANOVA, *, P < 0.05; **, P < 0.01; ***, P < 0.001).
Figure 1B, qRT-PCR of piwi-1 relative to GAPDH in RNAi animals as indicated, 2dpi. Circles represent individual biological replicates averaged from 3 technical replicates.D, Percentage of X1 cells from control and atm(RNAi) animals, 1dpi or unirradiated.
Figure 2E, qRT-PCR of piwi-1 relative to GAPDH in RNAi animals as indicated, 2dpi. Data are averages ± SD; circles represent biological replicates; statistics represent ordinary one-way ANOVA compared to controls, ****P<0.0001, ns=not significant.F, piwi-1 fluorescent in situ hybridization (FISH) of RNAi animals as indicated, 2 dpi.
Figure 3A, Top: Strategy for EdU pulse and animal fixation. Bottom: Confocal images of EdU-stained atm and control RNAi animals. B, Quantification by area of EdU+ cells from A. Circles represent individual animals. Inset, expanded scale showing compressed data below. Data includes two independent experiments.C, Graph showing percentage of G2/M cells in RNAi animals on indicated dpi.D, Confocal images of phosphohistone H3 (pH3)-stained RNAi animals after radiation exposure. Blue, DAPI. E and F, Quantification by area of pH3 cells in radiated (E) or unirradiated (F) animals after atm (solid line) and control (dashed line) RNAi.
Figure 4A, Confocal images of piwi-1 FISH in RNAi animals after radiation exposure. Scale bars=250µm. B and C, Percentage of animal area occupied by piwi-1 FISH divided by total animal area, in radiated (B) or unirradiated (C) RNAi animals. D, Total number of piwi-1+ cells in RNAi animals, 7 dpi. Experiments in (A) - (D) were repeated twice.E, Schematic of experimental design. Below, Kaplan-Meier survival curves of intact (solid line) and decapitated (dashed line) animals after RNAi as indicated. n = 45 animals, repeated twice.
Figure 5A - C, Kaplan-Meier survival curves of intact RNAi animals after radiation, n = 50 animals, repeated twice. Experiments in A and B were performed simultaneously but split into two graphs for clarity; control and atm(RNAi) values are replicated in both panels.D, Confocal images of piwi-1 FISH in RNAi animals after exposure to 20Gy, 2dpi. Scale bars=250µm. E, Quantification by area of piwi-1+ cells in RNAi animals after 20Gy exposure, 7dpi.
Figure 1A Western blot analysis of YTHDC1 in HEK293T and MCF-7 knockdown samples.B Volcano plot of genes with significant switching to lengthened (red) or shortened (blue) 3' UTRs according to test of linear trend alternative to independence in HEK293T (left panel) and MCF-7 cells (right panel). Embedded bar plots show the number of significant genes. The X axis shows the Pearson correlation r from the linear trend alternative test, and the y axis shows the FDR.E The qRT-PCR validation of APA switching. Eight genes with lengthened and shortened 3' UTRs in HEK293T and in MCF-7 cells were chosen. PRPF4B, CTNNBIP1, PHB and RCL1 were selected from HEK293T cells, and rest genes are MCF-7 cells. KD (Extended/Common)/Ctr (Extended/Common) <1 indicates a shorter 3' UTR in the knockdown sample, and vice versa. Data are presented as mean ± SEM of three biological replicates. *p<0.05, **p<0.01, the p values were obtained from unpaired two-tailed Student's t-test.
Figure 2A Higher proportion of target genes in the list of genes with shortened 3'UTRs after knockdown of YTHDC1. The p values were obtained by Fisher's exact test. *p=1.227×10-3, **p=3.725×10-4, NA: no significance.C Peak density distribution of YTHDC1 binding sites near different poly(A) sites. Genes with shortened 3' UTR shows a higher density of YTHDC1 binding peaks near proximal APA sites compared to background genes. The density of YTHDC1 binding site peak were calculated as the number of peak in a 10-nt interval divided by the total number of mRNAs that contained this position.E Scatter plot of protein and mRNA ratios of dual luciferase report assay with λN-BoxB system. λN, YTHDC1 and λN-YTHDC1 were co-transfected with a bicistronic vector. Two batches of experiments with three biological repeats were performed. Both luciferase activity and mRNA levels were measured. To test the effect of YTHDC1 binding on the proximal APA site, a linear model with the co-transfected genes and batch as independent variables was fitted. Data are presented as mean ± SEM of three biological replicates. ** p<0.01 with t-test from linear model.F Overexpression of YTHDC1-WT and YTHDC1-Mut (W377A, W428A) revealed by Western blot.G Scatter plot of qRT-PCR for APA validation. Mutation of the m6A binding site of YTHDC1 loses its inhibitory effect on proximal APA sites. Extended/Common ratios of ten genes were obtained by qRT-PCR in HEK293T cells overexpressing YTHDC1-WT and YTHDC1-Mut. The ratios were normalized to those of control cells. Data are presented as mean ± SEM of three biological replicates. *p<0.05, **p<0.01, ns: no significance. the p values were obtained from unpaired two-tailed Student's t-test.
Figure 3A-C The Co-IP results of endogenous WTAP, METTL3 and METTL14 with YTHDC1. The endogenous WTAP and YTHDC1 can be pulled down reciprocally by each other in the presence of RNase A.D The Co-IP results of Myc-CBLL1, Myc-RBM15, Myc-ZC3H13 and Myc-VIRMA with Endogenous YTHDC1. None of them can pull down Endogenous YTHDC1 in the presence of RNase A.E. Endogenous YTHDC1 interacts with RPB3 in a RNA independent way.
Figure 4A Co-IP assay using Myc-tagged 3' end processing factors to pull down endogenous YTHDC1 without the addition of RNase A. FIP1L1, PABPC1 and PABPC4 can pull down endogenous YTHDC1.B Co-IP assay using Myc-FIP1L1, Myc-PABPC1 and Myc-PABPC4 to pull down endogenous YTHDC1 in the presence of RNase A.C Co-IP assay with antibodies against endogenous YTHDC1 and FIP1L1.D Coomassie brilliant blue staining of purified different epitope-tagged YTHDC1 and FIP1L1.E An in vitro pull-down assay using purified FLAG-YTHDC1 and Myc-FIP1L1 showed that FLAG-YTHDC1 directly interacts with Myc-FIP1L1.F Endogenous YTHDC1 and FIP1L1 were stained with antibodies labeled with Alexa Fluor 488 and Alexa Fluor 568 fluorophores, respectively. Immunofluorescence results show that YTHDC1 and FIP1L1 have a high spatial association in the nucleus. Scale bars 15µm.G Scan graph analysis and nuclear staining density quantification of YTHDC1 and FIP1L1, indicating high colocalization in the nucleus. H Forty cells were randomly selected, and colocalization was assessed by calculating the average overlap coefficient, Pearson's correlation and colocalization rate according to Leica TCS SP8 microscope software. Data are presented as mean ± SD of 40 biological replicates.
Figure 5B Co-IP assays using truncated FIP1L1 proteins revealed that the proline-rich domain of FIP1L1 plays an important role in interacting with YTHDC1.C Mutation of FIP1L1 nearly abrogates the interaction between YTHDC1 and FIP1L1 compared with wild type.D-G Co-IP assays using different domains of YTHDC1 revealed that the N-terminus (1-344 aa) and YTH domain (364-507 aa) of YTHDC1 interact with FIP1L1.
Figure 6B YTHDC1 inhibits the interaction between Myc-FIP1L1 and FLAG-CPSF4. A substantially suppressed level of interaction between Myc-FIP1L1 and FLAG-CPSF4 was observed when FLAG-YTHDC1 input was increased.C Knockdown of YTHDC1 enhances endogenous FIP1L1 recruitment to CPSF4, indicating that YTHDC1 plays an important role in interfering with the 3' end processing complex interaction.D YTHDC1 has little effect on the interaction between Myc-FIP1L1 and FLAG-PAPOLA. The interaction between Myc-FIP1L1 and FLAG-PAPOLA was not significantly affected when FLAG-YTHDC1 was increased.E Mutation of proline had little effect on the interaction between FIP1L1 and CPSF4 compared with the wild type.F The mutation of prolines in FIP1L1 abrogated the inhibitory effect of YTHDC1 on the interaction between FIP1L1 and CPSF4.G qRT-PCR validation of APA site switching in a bicistronic dual luciferase system. FIP1L1-Mut could significantly increase the ratios of Rluc/Fluc compared to FIP1L1-WT, indicating that YTHDC1 inhibits the use of proximal APA sites by interacting with the proline-rich domain of FIP1L1. Data are presented as mean ± SEM of three biological replicates. **p=8.77×10-4, the p values were obtained with unpaired two-tailed Student's t-test.
Figure 7B Microscopy images of fluorescence recovery following partial photobleaching of GFP-FIP1L1. Normal solutions containing 2.1 mg/ml GFP-FIP1L1 were examined with a microscope at different time points. Scale bars: upper 3 µm, lower 2.98 µm.C FRAP assay of GFP-FIP1L1. Quantification of average relative fluorescence intensity and its initial rate of area of photobleaching across 8 individual GFP-FIP1L1 droplets. Data are presented as mean ± SD of 8 biological replicates.D The GFP-FIP1L1 phase separation assay was performed in normal buffer, high salt buffer and normal buffer with the addition of RNA. All these results indicated that the GFP-FIP1L1 protein displayed dynamic and liquid-like properties. Scale bars 15µm.E Sanger sequencing was used to genotype eGFP-FIP1L1 and mScarlet-YTHDC1 homozygous cell lines.F In vivo FRAP assays for eGFP-FIP1L1 and DsRed-YTHDC1 were examined by time-lapse phase-contrast imaging. The arrows represent droplet of GFP-FIP1L1.Nine cell-independent experiments were selected for FRAP assays. Scale bars 10 µm. G, Quantification of average relative fluorescence intensity and its initial rate of area of photobleaching across 15 individual GFP-FIP1L1 droplets in figure G respectively. Data are presented as mean ± SD of 15 biological replicates.H Quantification of average relative fluorescence intensity and its initial rate of area of photobleaching across 15 individual DsRed-YTHDC1 in figure H, respectively. Data are presented as mean ± SD of 15 biological replicates.I Genome-edited eGFP-FIP1L1 HEK293T cells expressing Dsred-YTHDC1-WT and Dsred-YTHDC1-Mut were examined by confocal microscopy. DsRed-YTHDC1-WT forms LLPS to compartmentalize FIL1L1, but DsRed-YTHDC1-Mut diffuses in the nucleus without droplet structure. Droplet structures are indicated by arrows. Scale bars: upper 8.09 µm, lower 6.64 µm. J Colocalization of YTHDC1-WT and YTHDC1-Mut in figure I was assessed by calculating the average overlap coefficient according to Leica TCS SP8 microscope software. Data are presented as mean ± SEM of 15 cells. ****p=6.46×10-4, the p values were obtained with unpaired two-tailed Student's t-test.
Figure 1D. Validation of selected CRISPR screening hits by targeted gene inactivation. Genes are plotted on the x-axis from lowest FDR value (IFIT1) to highest FDR value (CD163L1) as determined by MAGeCK analysis of the genome-wide screen. All samples were normalized to non-targeting controls ("Non-Targ") for graphing. Graphs represent the mean with error bars representing standard deviation. One-way ANOVA with Dunnett's test comparing each group to the non-targeting control was performed on non-normalized data, n=3 biological replicates.
Figure 2D. Heat maps of FPKM (Left) and log2FC (Right) values (from RNA-Seq) of IFN-induced CRISPR screening hits at 6 and 24 h post IFN-treatment. Heatmap intensity represents the mean of 3 independent biological replicates.E. Western Blots from lysates of U-2 OS cells treated with IFNβ (20 U/mL) for 6 and 24 h. Antibodies were used to probe for IFN-induced CRISPR screening hits.
Figure 3A. Effects of CRISPR-based gene inactivation of IRF9, STAT1, and STAT2 or ZC3HAV1, IFIT3, and IFIT1 on VEEV infection (MOI of 10 for 5 h) in U-2 OS cells with IFNβ pretreatment (20 U/mL for 24 h). B. Effects of CRISPR-based gene inactivation of IRF9, STAT1, and STAT2 or ZC3HAV1, IFIT3, and IFIT1 on VEEV infection (MOI of 10 for 4.5 h) in Huh7.5 cells with (+ symbols) and without (- symbols) IFNβ pretreatment (100 U/mL for 24 h). Data information: data are presented as mean +/- standard deviation. n=3 independent biological replicates, one-way ANOVA with Dunnett's test. ns p > 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.D. Dose response curve of IFNβ pretreatment (Left, 2-fold dilutions from 200 U/mL to 1.6 U/mL for 24 h) or IFNλ pretreatment (Right, 2-fold dilutions from 500 ng/mL to 3.9 ng/mL for 24 h) demonstrating inhibition of VEEV infection (MOI of 5 for 5 h) in control (circles), IRF9/STAT1/STAT2-targeted (squares), or ZAP/IFIT3/IFIT1-targeted (triangles) U-2 OS cells. Graphs represent the mean of normalized and transformed (from %Infection to %Inhibition) data for 3 biological replicates with error bars representing standard deviation.
Figure 4A. U-2 OS control, IRF9/STAT1/STAT2-targeted, and ZAP/IFIT3/IFIT1-targeted cells were infected with GFP-expressing ONNV virus (left) or ZIKV (right) after IFNβ pretreatment (20 U/mL). Data information: In data are presented as mean +/- standard deviation. n=3 independent biological replicates, one-way ANOVA with Dunnett's test. ns p > 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.B. Huh7.5 control, IRF9/STAT1/STAT2-targeted, and ZAP/IFIT3/IFIT1-targeted cells were infected with GFP-expressing Sindbis virus (AR86 strain, left) or vesicular stomatitis virus (right) after IFNβ pretreatment (100 U/mL). The percentage of infected (GFP+) cells was quantified by flow cytometry. Data information: In data are presented as mean +/- standard deviation. n=3 independent biological replicates, one-way ANOVA with Dunnett's test. ns p > 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Figure 5A. Heatmaps showing the ratio of infected cells at 6, 9, and 12 h of infection with VEEV-GFP. U-2 OS control, IRF9/STAT1/STAT2-targeted, or ZAP/IFIT3/IFIT1-targeted cells were treated with or without IFNβ (20 U/mL for 24 h) and infected with an MOI of 0.5 (left), 1 (middle), or 5 (right) of VEEV-GFP. Infection was quantified by flow cytometry. Heatmap intensity represents the mean of 3 independent biological replicates.B. (Top) Quantification of VEEV production (12 h post-infection, MOI of 2, titered in BHK-21J cells) by plaque assay of control (far left: without IFN-pretreatment), IRF9/STAT1/STAT2-targeted, or ZAP/IFIT3/IFIT1-targeted cells after IFNβ pretreatment (IFNβ 20 U/mL for 24 h). (Bottom) Western blot shows IFN-mediated induction and CRISPR-based targeting of IRF9, STAT1, STAT2, ZAP, IFIT3, and IFIT1. Red asterisks denote long (**) and short (*) isoform of ZAP. Data information: data are presented as mean +/- standard deviation. n=3 independent biological replicates, one-way ANOVA with Dunnett's test on log transformed data. ns p > 0.05, ***p < 0.001, and ****p < 0.0001.C. (Top) Quantification of VEEV production (12 h post-infection, MOI of 2, titered in BHK-21J cells) by plaque assay in control (far left: without IFN-pretreatment), IRF9/STAT1/STAT2-targeted, ZAP/IFIT3/IFIT1-targeted, or ZAP/IFIT3/IFIT1-targeted cells that were subsequently CRISPR targeted with a guide targeting BST2, RSAD2, ISG20, or IFITM3 after IFNβ pretreatment (IFNβ 20 U/mL for 24 h). (Bottom) Western blot shows IFN-mediated induction and CRISPR-based targeting of BST2, RSAD2, ISG20, and IFITM3. Black squares denote non-specific bands. Data information data are presented as mean +/- standard deviation. n=3 independent biological replicates, one-way ANOVA with Dunnett's test on log transformed data. ns p > 0.05, ***p < 0.001, and ****p < 0.0001.
Figure 1(A) Drosophila egg chambers are composed of a large oocyte surrounded by a columnar follicle cell epithelium that activates a Hpo kinase bimolecular fluorescence complementation (BiFC) dimerization sensor (HpoKD-Venus; green) apically in small punctae. Note that the Hpo sensor is activated in columnar cells but not in mechanically stretched cells. (B) Nutrient restriction (starvation) of adult females for 24h causes formation of large micron-scale Hpo sensor punctae.(D) Inhibition of PI3K by overexpression of a dominant-negative version (the p60 subunit alone) is sufficient to induce formation of large micron-scale Hpo sensor punctae, while activation of Akt by overexpression of a myristylated form of the protein is sufficient to reduce formation of the small apical Hpo sensor punctae that are normally observed in control animals.
Figure 2(A) Drosophila egg chambers at stages 9/10 of oogenesis expressing a Hpo kinase dimerization sensor that forms small punctae at the apical domain of the follicle cell epithelium that surrounds the oocyte. (B) Treatment of egg chambers with 1,6-hexanediol (10%) for 5 min abolishes formation of Hpo sensor punctae. (C) Treatment of egg chambers with 1M Sorbitol to osmotically withdraw water, and thereby induce macromolecular crowding, is sufficient to induce formation of large micron-scale Hpo sensor punctae. (D) Nutrient restriction (starvation) of adult females for 24h induces large micron-scale Hpo sensor punctae that are similar in size to those in (C). (E) Treatment of egg chambers from adult females starved for 24h with 1,6-hexanediol (10%) for 5 min abolishes formation of Hpo sensor punctae.(I) Quantification of FRAP recovery as a fraction of the initial fluorescence intensity. The rapid initial slope of recovery suggests dynamic exchange between the condensate and the cytoplasmic pool of the proteins. Temporal averages are plotted as individual data points (n>3 biological replicates), error bars represent one standard deviation from the mean.
Figure 3(A) Apical cross-sections of Drosophila ovarian follicle cell epithelia at high magnification, showing accumulation of Hpo-YFP punctae at the apical domain, which is strongly enhanced upon 24h starvation to reduce Insulin/IGF-1 signalling. Hpo puncta are lost following a short treatment with 1,6-hexanediol. Samples were fixed before imaging. (B) Quantification of Hpo-YFP puncta number and size in (A). Biological replicates are plotted as individual data points (n>13 for all samples), error bars represent one standard deviation from the mean, statistical significance was determined using a t-test ****P<0.0001 ***P<0.001 **P<0.01 *P<0.05.(C) Apical cross-sections of Drosophila ovarian follicle cell epithelia at high magnification, showing loss of Hpo-YFP punctae upon mechanical strain in 'stretch cells'. The Drosophila follicle cell epithelium at stage 10 is comprised of anterior stretch cells (flat) and columnar cells. Endogenously-tagged Hpo punctae are strongly enriched at the apical domain of columnar cells but not in flattened stretch cells. White asterisks demarcate nuclei of individual stretch cells. (D) Quantification of Hpo-YFP total intensity, and size in (C). Biological replicates are plotted as individual data points (n>10 for all samples), error bars represent one standard deviation from the mean, statistical significance was determined using a t-test ****P<0.0001 ***P<0.001 **P<0.01 *P<0.05.
Figure 4(A) Expanded (Ex; endogenously tagged with YFP) localises to the apical domain of columnar follicle cells at stages 9/10 of oogenesis. (B) The Hpo dimerization sensor localises in a similar pattern to Ex.(E) Condensates induced by high levels of Ex in the cytoplasm co-localise with the Hpo sensor but do not co-localise with Crb, which remains apical. (F) Condensates induced by high levels of Ex in the cytoplasm immunostain positively for phosphorylated Wts.(G) Moderate overexpression of untagged Kib is sufficient to induce formation of large micron-scale HpoKD-Venus condensates that primarily localise apically. DAPI marks nuclei (blue). (H) Overexpression of Kib-GFP with the Gal4/UAS system is sufficient to induce formation of large micron-scale cytoplasmic condensates.
Figure 5(A) Human U2OS cells in culture immunostained for endogenous Hippo pathway components reveals punctate subcellular localisation within the cytoplasm.(B) Ectopic expression of Flag-tagged human Kibra (KIB-Flag) generates large spherical punctae in the cytoplasm of human Caco2 epithelial cells (top). Co-localisation of KIB-GFP with active phosphorylated LATS (pLATS) in the cytoplasm of human Caco2 epithelial cells (middle). Note that cells expressing KIB-GFP have increased Hippo signalling as indicated by reduced levels of nuclear YAP localisation (bottom). Dashed lines demarcate transfected cells.
Figure 6(A) Expression of KIB-GFP, KIB∆C-GFP or KIB∆M+C-GFP in human HEK293T cells and co-staining for pLATS. DAPI marks nuclei in blue.
Figure 7(A) Human Caco2 intestinal epithelial cells are relatively flat when cultured as cell lines in 2D (Caco-2 cells, top, middle) and cuboidal when cultured in 3D (spheroid, bottom). Punctae of pLATS (green) are strongly enriched at the apical domain of cuboidal cells grown in 3D culture but not in flattened cells grown in 2D culture. Note the increased apical enrichment in medium-density culture in 2D, where cells become more cuboidal than at low-density. Phalloidin staining marks the actin cytoskeleton in red. DAPI marks nuclei in blue.(C) Mouse small intestinal organoids can become stretched upon growth as inflated spheres, which leads to mechanical strain in epithelial cells and reduced pLATS localisation apically, which correlates with reduced Hippo signalling (leading to increased YAP nuclear localisation). White arrows highlight the apical domain of epithelial cells.
Figure 8(B) High resolution imaging of intestinal organoids cultured in control medium or medium supplemented with the NRG1 growth factor for 1 h which strongly stimulates pAkt, reduces pLATS staining, and drives YAP to the nucleus.
Figure 1(D) The 43 overlapping candidate genes are described in Figure 1C. Known driver genes listed in the Cancer Gene Census (CGC) or Oncology Knowledge Base (OncoKB) were removed from further analysis. Genes were assessed for a known association with the DDR pathway.(H) Immunofluorescence staining of γ.H2.AX expression of cells described in Figure 1G.
Figure 2(D) Enrichment of control-sgRNA (n=5) and Slf2-sgRNA (n=6) transduced HSPCs over time.(E) Kaplan-Meier curves of overall survival of mice transplanted with Eµ-myc;Rosa26Cas9 HSPCs transduced with control-sgRNA (n=15) or sgRNA targeting Slf2 (n=6). p < 0.0001, log-rank (Mantel-Cox) test.(F) Immunohistochemical analysis of Slf2-sgRNA lymphomas described in Figure 2E. Representative example of three analyzed Slf2-sgRNA lymphomas.
Figure 3(B, C) Kaplan-Meier overall survival curves of the indicated cohorts of DLBCL patients (Lenz, NEJM). DLBCL patients were classified according to SLF2 or SLF1 mRNA expression. P-value was determined by log-rank (Mantel-Cox) test.(E) Mafosphamide dose-response curves of SLF2KO and control SU-DHL-5 cells. Cells were treated with mafosphamide (MAF; 0, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000 ng/ml) for 48 hours and viability was determined by DAPI staining and flow cytometry measurement. Error bars represent SD from three independent experiments.(G) Representative plots of the flow cytometry experiment described in Figure 3F.
Figure 4(A) Immunoblot analysis of U-2-OS cells following CRISPR/Cas9 mediated knockout of SLF2 upon DRB treatment (0.5 µM) for indicated times with the indicated antibodies.(B) Immunoblot analysis of a panel of human DLBCL cell lines with a specific SLF2 antibody and quantification of the SLF2 western blots (middle panel). Protein expression of SLF2 was normalized to β-Actin expression. The expression level of SLF2 in DB cells was set as 1. Data are presented as mean +/- SD of n=3 independent experiments.(E) Immunoblot analysis of human SU-DHL-5 control and SLF2KO cell lines upon DRB treatment (0.5 µM) for indicated time points with the indicated antibodies.