The Ewing Sarcoma Protein Regulates DNA Damage-Induced Alternative Splicing  Maria Paola Paronetto, Belén Miñana, Juan Valcárcel  Molecular Cell  Volume 43, Issue 3, Pages 353-368 (August 2011) DOI: 10.1016/j.molcel.2011.05.035 Copyright © 2011 Elsevier Inc. Terms and Conditions

Molecular Cell 2011 43, 353-368DOI: (10.1016/j.molcel.2011.05.035) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 Alternative Splicing Changes Induced by EWS Knockdown (A) Reduced levels of EWS protein upon RNAi of EWS in HeLa cells. Western blot analysis of the indicated amounts of extracts from cells transfected with either scrambled or siEWS oligonucleotides, prepared 72 hr after transfection. PVDF membranes were hybridized with EWS or ERK2 antibodies, as indicated. Quantification of EWS western blot signals is shown; error bars represent standard deviation of the quantification of six independent experiments. (B) AS categories affected by EWS knockdown. The fraction of AS events in each of five different AS classes is indicated for all the events monitored in the array (black) and for those affected by EWS knockdown (gray). (C) Gene Ontology (GO, Ingenuity Pathway) analysis of genes with AS events affected by EWS knockdown. Blue bars indicate the number of genes in a particular pathway present among those harboring AS changes induced by EWS knockdown. The orange line links values of GO enrichment for each pathway, obtained as ratios between the number of genes in the pathway affected by EWS depletion and the total number of genes in that pathway. The threshold for statistically significant (p < 0.05) enrichment is 1.3. (D–G) RT-qPCR validation of microarray-predicted AS changes in the indicated genes. AS patterns are schematized in the upper part of each panel. (C), (D), and (E) represent exon-junction probes monitored in the microarray and also used for qPCR. (B) represents probes within the alternative exon. Histograms represent the ratio between qPCR amplification signals characteristic of each isoform, obtained from RNAs isolated from cells transfected with siEWS versus scrambled oligonucleotides (pale gray bars) or from cells stably transfected with pLKOshEWS versus pLKO vectors (dark gray bars). Ratios of AS amplification products were then normalized to the ratios corresponding to a constitutive exon (GE). Average and standard deviation for three independent biological replicas are shown. p values for gene/isoform expression changes were <0.02 and in most cases <0.001. (H) Western blot analysis of extracts from HeLa cells transfected with scrambled or siEWS oligonucleotides. Extracts (10 μg) were loaded in each lane, and the membranes were hybridized with antibodies specific for the indicated proteins. See also Figures S1 and S2. Molecular Cell 2011 43, 353-368DOI: (10.1016/j.molcel.2011.05.035) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 EWS Associates Directly with Its RNA Targets (A) RT-qPCR analysis of RNA precipitates from total RNA purified from HeLa cells pulled down by GST-EWS or GST. The histograms represent the ratio between signals associated with GST-EWS versus GST pellets, normalized by the values obtained for GAPDH mRNA. Average and standard deviations for three independent experiments are represented. (B–E) Electrophoretic mobility shift assays (EMSA) using purified GST-EWSCter (concentrations of 10–100 nM) and RNA probes corresponding to CHEK2 exons 2 (B) and 3 (E), MAP4K2 exon 9 (C), ABL1 exon 3 (D), and QKI exon 2 (E). The positions of free RNA and RNA-protein complexes are indicated. (F–I) RT-qPCR quantification of the ratio between RNA levels present in CLIP precipitates using anti-EWS antibodies (gray bars) or control IgGs (black bars), using primer pairs covering intron-exon boundaries of CHEK2, MAP4K2, ABL1, and QKI pre-mRNAs, as represented in the scheme of the genomic regions involved. Alternatively spliced exonic regions are indicated in white. Histograms represent the average and standard deviations of the fraction of input detected in the EWS precipitates of three independent experiments, normalized to IgGs precipitates. See also Figure S3. Molecular Cell 2011 43, 353-368DOI: (10.1016/j.molcel.2011.05.035) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 Association of EWS with Chromatin at Genomic Regions Harboring EWS-Mediated AS Changes (A–D) qPCR quantification of the ratio between the levels of genomic DNA present in chromatin immunoprecipitates (ChIP) using antibodies against EWS (gray bars) versus control IgGs (black bars), using primers covering intron-exon boundaries of CHEK2, MAP4K2, ABL1, and QKI, schematized as in Figure 2. Histograms represent the average and standard deviations of the fraction of input detected in the EWS precipitates of three independent experiments, normalized to IgGs precipitates. See also Figures S4 and S5. Molecular Cell 2011 43, 353-368DOI: (10.1016/j.molcel.2011.05.035) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 UV Irradiation Leads to AS Changes Similar to Those Induced by EWS Knockdown (A–C) RT-qPCR analyses of RNA extracted from HeLa cells irradiated with 10 J/m2 UV light and harvested 6 hr after irradiation. Histograms represent the ratio of qPCR amplification signals for each isoform of CHEK2, ABL1, and MAP4K2 AS regions (as in Figure 1) between irradiated and nonirradiated cells, normalized to the ratios corresponding to a constitutive exon (GE). Error bars represent standard deviations of three independent experiments. (D) Design of BrU labeling and UV irradiation experiments. Labeling was performed by exposure of cells to 2 mM BrU for 1 hr, either before, immediately after, or 2 or 5 hr after UV light irradiation. (E and F) RT-qPCR analysis of BrU-labeled RNAs (precipitated with anti BrU antibodies) at different times after UV light irradiation (1, 3, and 6 hr), as indicated in (D). Histograms represent average and standard deviation (n = 3) of the ratio of amplification products monitoring probe E (corresponding to skipping of MAP4K2 exon 9 or alternative splice site donor in ABL1 exon 3) in irradiated versus nonirradiated cells, normalized to gene expression in control nonirradiated cells. (G) Western blot analysis of total extracts from HeLa cells irradiated with 10 J/m2 and harvested 1, 3, or 6 hr after treatment, using antibodies against α-Ph-ERKs, α-PhJNKs, α-Ph-CHK1, α-Ph-CHK2, α-Ph-p38 (as controls of activation of DNA damage response), α-CHK2, α-EWS, α-c-ABL, and α-αtubulin antibodies. Of total HeLa extracts, 10 μg was loaded in each lane. Molecular Cell 2011 43, 353-368DOI: (10.1016/j.molcel.2011.05.035) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 EWS Translocates into the Nucleolar Compartment upon UV Irradiation (A) Immunofluorescence analysis of HeLa cells treated with 10 J/m2 UV light and harvested at 30′, 60′, 3 hr, 5 hr, and 6 hr after UV light treatment. Cells were stained for EWS (rabbit anti-EWS dilution 1:1000) and DNA (Hoechst). (B) Immunofluorescence analysis of HeLa cells treated with 10 J/m2 UV light. Cells were fixed 6 hr after UV light treatment and stained for hnRNP A1 (mouse α-hnRNP A1, Sigma, dilution 1:500), EWS (rabbit anti-EWS, dilution 1:1000), and DNA (Hoechst). (C) Immunofluorescence analysis of HeLa cells transfected with GFP-EWS and irradiated with UV light (10 J/m2). Cells were fixed 6 hr after UV light treatment and visualized for GFP fluorescence and stained for nucleolin (mouse α-NCL, Abcam, dilution 1:1000) and DNA (Hoechst). See also Figure S6. Molecular Cell 2011 43, 353-368DOI: (10.1016/j.molcel.2011.05.035) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 Weakened Interaction of EWS with Its Splicing Targets upon UV Irradiation (A–C) qPCR analysis of EWS ChIP signals, carried out as in Figure 3, for control- and UV-irradiated cells. Histograms represent average and standard deviation values for three independent experiments. (D–F) qPCR analysis of EWS CLIP signals, carried out as in Figure 2, for control- and UV-irradiated cells. Histograms represent average and standard deviation values for three independent experiments. Molecular Cell 2011 43, 353-368DOI: (10.1016/j.molcel.2011.05.035) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 Time Course of EWS Translocation and AS Changes and Requirement of EWS for Efficient Response to Genotoxic Stress (A) Immunofluorescence analysis of HeLa cells treated with UV light (10 J/m2) and harvested at different time points after the treatment (6, 12, 24, 36, 48 hr). Cells were stained with mouse α-SC35 (red) and rabbit α-EWS (green) antibodies, and DNA was stained with Hoechst (blue). (B) Semiquantitative RT-PCR analysis of cDNA from HeLa cells treated with 10 J/m2 UV light and harvested at different time points after the treatment (6, 12, 24, 36, 48 hr) using primers amplifying MAP4K2 transcripts from exons 8–12. The histogram represents average and standard deviations of the ratio between exon skipping and inclusion for three independent experiments. (C–E) RT-qPCR analysis as in Figure 4 of RNAs isolated from HeLa cells treated with 10J/m2 UV light and harvested at different time points after the treatment (6, 12, 24, 36, 48 hr). Histograms represent average and standard deviation values for three independent experiments. (F) Quantification of clonogenic assays using HeLa cells stably infected with pLKO or pLKO-shEWS vectors, irradiated with different doses of UV light, as indicated, and harvested 10 days after treatment. Histograms represent average and standard deviations of three independent assays. Gray bars represent values for pLKO (control)-infected cells, while black bars represent values for pLKO-shEWS-infected cell lines. (G) Expression of c-ABL restores the clonogenic potential of EWS knockdown cells upon irradiation with UV light. Clonogenic assays as in (F) were carried out in cells transfected with a plasmid expressing c-ABL or with control vector. Histograms represent average and standard deviations for three independent assays. (H) Viability assay of HeLa cells stably infected with pLKO or pLKO-shEWS vectors, treated with UV light (10 J/m2) and harvested 72 hr after the treatment, with or without a second hit of UV light at 12, 24, and 48 hr after the first hit. Ten thousand cells per sample were stained with Annexin V and PI, and cell viability was analyzed using a FACS Canto cell sorter. Histograms represent average and standard deviations of three independent viability assays. Gray bars represent values for pLKO, dark bars represent values for pLKO-shEWS infected cell lines. See also Figure S7. Molecular Cell 2011 43, 353-368DOI: (10.1016/j.molcel.2011.05.035) Copyright © 2011 Elsevier Inc. Terms and Conditions