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• W2952073075 abstract "Article21 June 2019Open Access Source DataTransparent process Arginine methylation of the DDX5 helicase RGG/RG motif by PRMT5 regulates resolution of RNA:DNA hybrids Sofiane Y Mersaoui Sofiane Y Mersaoui orcid.org/0000-0001-6905-3600 Departments of Oncology and Medicine, Segal Cancer Center, Lady Davis Institute for Medical Research, McGill University, Montréal, QC, Canada Search for more papers by this author Zhenbao Yu Zhenbao Yu orcid.org/0000-0002-2583-6854 Departments of Oncology and Medicine, Segal Cancer Center, Lady Davis Institute for Medical Research, McGill University, Montréal, QC, Canada Search for more papers by this author Yan Coulombe Yan Coulombe Genome Stability Laboratory, Oncology Division, CHU de Québec-Université Laval, Québec, QC, Canada Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University Cancer Research Center, Québec, QC, Canada Search for more papers by this author Martin Karam Martin Karam Departments of Oncology and Medicine, Segal Cancer Center, Lady Davis Institute for Medical Research, McGill University, Montréal, QC, Canada Search for more papers by this author Franciele F Busatto Franciele F Busatto Genome Stability Laboratory, Oncology Division, CHU de Québec-Université Laval, Québec, QC, Canada Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University Cancer Research Center, Québec, QC, Canada Search for more papers by this author Jean-Yves Masson Corresponding Author Jean-Yves Masson [email protected] orcid.org/0000-0002-4403-7169 Genome Stability Laboratory, Oncology Division, CHU de Québec-Université Laval, Québec, QC, Canada Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University Cancer Research Center, Québec, QC, Canada Search for more papers by this author Stéphane Richard Corresponding Author Stéphane Richard [email protected] orcid.org/0000-0003-2665-4806 Departments of Oncology and Medicine, Segal Cancer Center, Lady Davis Institute for Medical Research, McGill University, Montréal, QC, Canada Search for more papers by this author Sofiane Y Mersaoui Sofiane Y Mersaoui orcid.org/0000-0001-6905-3600 Departments of Oncology and Medicine, Segal Cancer Center, Lady Davis Institute for Medical Research, McGill University, Montréal, QC, Canada Search for more papers by this author Zhenbao Yu Zhenbao Yu orcid.org/0000-0002-2583-6854 Departments of Oncology and Medicine, Segal Cancer Center, Lady Davis Institute for Medical Research, McGill University, Montréal, QC, Canada Search for more papers by this author Yan Coulombe Yan Coulombe Genome Stability Laboratory, Oncology Division, CHU de Québec-Université Laval, Québec, QC, Canada Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University Cancer Research Center, Québec, QC, Canada Search for more papers by this author Martin Karam Martin Karam Departments of Oncology and Medicine, Segal Cancer Center, Lady Davis Institute for Medical Research, McGill University, Montréal, QC, Canada Search for more papers by this author Franciele F Busatto Franciele F Busatto Genome Stability Laboratory, Oncology Division, CHU de Québec-Université Laval, Québec, QC, Canada Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University Cancer Research Center, Québec, QC, Canada Search for more papers by this author Jean-Yves Masson Corresponding Author Jean-Yves Masson [email protected] orcid.org/0000-0002-4403-7169 Genome Stability Laboratory, Oncology Division, CHU de Québec-Université Laval, Québec, QC, Canada Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University Cancer Research Center, Québec, QC, Canada Search for more papers by this author Stéphane Richard Corresponding Author Stéphane Richard [email protected] orcid.org/0000-0003-2665-4806 Departments of Oncology and Medicine, Segal Cancer Center, Lady Davis Institute for Medical Research, McGill University, Montréal, QC, Canada Search for more papers by this author Author Information Sofiane Y Mersaoui1,‡, Zhenbao Yu1,‡, Yan Coulombe2,3, Martin Karam1, Franciele F Busatto2,3, Jean-Yves Masson *,2,3 and Stéphane Richard *,1 1Departments of Oncology and Medicine, Segal Cancer Center, Lady Davis Institute for Medical Research, McGill University, Montréal, QC, Canada 2Genome Stability Laboratory, Oncology Division, CHU de Québec-Université Laval, Québec, QC, Canada 3Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University Cancer Research Center, Québec, QC, Canada ‡These authors contributed equally to this work *Corresponding author. Tel: +1 418 525 4444 x 15154; E-mail: [email protected] *Corresponding author. Tel: +1 514 340 8260; E-mail: [email protected] The EMBO Journal (2019)38:e100986https://doi.org/10.15252/embj.2018100986 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Aberrant transcription-associated RNA:DNA hybrid (R-loop) formation often causes catastrophic conflicts during replication, resulting in DNA double-strand breaks and genomic instability. Preventing such conflicts requires hybrid dissolution by helicases and/or RNase H. Little is known about how such helicases are regulated. Herein, we identify DDX5, an RGG/RG motif-containing DEAD-box family RNA helicase, as crucial player in R-loop resolution. In vitro, recombinant DDX5 resolves R-loops in an ATP-dependent manner, leading to R-loop degradation by the XRN2 exoribonuclease. DDX5-deficient cells accumulate R-loops at loci with propensity to form such structures based on RNA:DNA immunoprecipitation (DRIP)-qPCR, causing spontaneous DNA double-strand breaks and hypersensitivity to replication stress. DDX5 associates with XRN2 and resolves R-loops at transcriptional termination regions downstream of poly(A) sites, to facilitate RNA polymerase II release associated with transcriptional termination. Protein arginine methyltransferase 5 (PRMT5) binds and methylates DDX5 at its RGG/RG motif. This motif is required for DDX5 interaction with XRN2 and repression of cellular R-loops, but not essential for DDX5 helicase enzymatic activity. PRMT5-deficient cells accumulate R-loops, resulting in increased formation of γH2AX foci. Our findings exemplify a mechanism by which an RNA helicase is modulated by arginine methylation to resolve R-loops, and its potential role in regulating transcription. Synopsis Association of the helicase DDX5 and the nuclease XRN2 is dependent on PRMT5-mediated arginine methylation of DDX5 and important for resolution of RNA/DNA hybrids at transcriptional termination regions, implicating this mechanism in XRN2-mediated transcriptional termination. PRMT5 inhibition leads to R-loops accumulation and DNA damage. Arginine methylation of the DDX5 RGG/RG motif regulates its association with XRN2 nuclease. DDX5 functions with XRN2 to resolve R-loops at transcription termination sites. Recombinant DDX5 resolves R-loops, but not D-loops, in vitro via its helicase activity. Introduction In mammalian cells, there are nine protein arginine methyltransferases (PRMTs) responsible for transferring methyl groups from S-adenosyl methionine to the nitrogen atoms of arginine (Bedford & Clarke, 2009). Arginine methylation modulates multiple biological processes from gene expression, pre-mRNA splicing, the DNA damage response, and signal transduction (Blanc & Richard, 2017). PRMT5 is the major enzyme catalyzing symmetrical dimethylarginines, and as such, multiple responses have been observed upon inhibition of its function including the induction of the p53 response, DNA damage, and cell death. PRMT5 regulates gene expression by methylating histones and transcription factors (Friesen et al, 2001; Pal et al, 2004; Cho et al, 2012). Methylation of the Sm proteins by PRMT5 regulates the p53 response by modulating the alternative splicing of MDM4 and MDM2 (Bezzi et al, 2013). Inhibition of arginine methylation has been shown to induce DNA damage and sensitize cells to DNA-damaging agents, reviewed in Blanc and Richard (2017). PRMT5 regulates TIP60 activity by RUVBL1 methylation (Clarke et al, 2017) and by TIP60 alternative splicing to regulate the DDR (Hamard et al, 2018). This multitude of responses triggered in the absence of PRMT5 function suggests that it may be a good therapeutic target. Indeed, PRMT5 inhibition has been shown to decrease tumor growth in mouse models of mantle cell lymphoma, AML, CML, B-cell lymphoma, glioma, and breast cancer (Chan-Penebre et al, 2015; Koh et al, 2015; Li et al, 2015; Zhou et al, 2016; Braun et al, 2017; Hamard et al, 2018; Kaushik et al, 2018; Tan et al, 2019). Physiologically, R-loops or RNA:DNA hybrids are programmed structures that occur during many cellular processes, including transcription, replication, and immunoglobulin class switching (Skourti-Stathaki & Proudfoot, 2014; Chedin, 2016; Bhatia et al, 2017). Persistent R-loops impede DNA replication and if unresolved ultimately cause DNA breaks and genomic instability (Hamperl & Cimprich, 2016; Aguilera & Gomez-Gonzalez, 2017) or mitochondrial instability (Silva et al, 2018). Therefore, it is not surprising that there are specialized machineries or protein complexes to prevent and resolve these R-loops. These structures can be unwound by RNA:DNA helicases, such as Senataxin (SETX) and Aquarius (AQR) (Skourti-Stathaki et al, 2011; Bhatia et al, 2014; Sollier et al, 2014; Hatchi et al, 2015), and the RNA in RNA:DNA hybrids degraded by RNase H1 and RNase H2 (Wahba et al, 2011). The RNA helicase Senataxin has been shown to resolve R-loops in vitro, and its deficiency in cells leads to R-loop accumulation (Skourti-Stathaki et al, 2011; Sollier et al, 2014; Hatchi et al, 2015). Senataxin is reported to function with the 5′–3′ exonuclease XRN2 to resolve a subset of R-loops at transcription termination sites of actively transcribed genes (Skourti-Stathaki et al, 2011; Morales et al, 2016; Aymard et al, 2017). The DNA helicase RECQ5 and RNA helicases DDX1 (Li et al, 2008, 2016; Ribeiro de Almeida et al, 2018), DDX19 (Hodroj et al, 2017), DDX21 (Song et al, 2017), DDX23 (Sridhara et al, 2017), and DHX9 (Cristini et al, 2018) were also found to be functionally involved in suppression of R-loops. Topoisomerase I removes the negative supercoils behind RNA polymerases to prevent annealing of the nascent RNA with the DNA template and suppresses R-loop formation (Tuduri et al, 2009). Fanconi anemia (FA) pathway proteins resolve RNA:DNA hybrids via FANCM translocase activity (García-Rubio et al, 2015; Schwab et al, 2015). Several RNA-processing proteins, such as the THO complex and the SRSF splicing factor, suppress R-loop formation, for example, by preventing the availability of the nascent RNAs for hybridization to template DNA (Huertas & Aguilera, 2003; Li & Manley, 2005; Paulsen et al, 2009; Wahba et al, 2011; Stirling et al, 2012; Sollier et al, 2014). The homologous recombination proteins, BRCA1 and BRCA2, are also involved in R-loop prevention and resolution (Bhatia et al, 2014; Hatchi et al, 2015). Predictably, mutations of proteins that prevent R-loop accumulation are frequently found in human diseases (Bhatia et al, 2017). Despite these findings, little is known of the post-translational modifications regulating R-loop formation and resolution. It has been shown that the pausing of RNA polymerase II (RNA Pol II) increases DDX23 phosphorylation by SRPK2 enhancing R-loop suppression (Sridhara et al, 2017). Acetylation of DDX21 by histone acetyltransferase CBP regulates its helicase activity (Song et al, 2017), while methylation of RNA Pol II subunit POLR2A by PRMT5 regulates Senataxin recruitment at transcription termination regions (Zhao et al, 2016). Moreover, methylation of TDRD3 by CARM1 regulates the recruitment of topoisomerase IIIB at the c-Myc locus preventing negative supercoiling and R-loops (Yang et al, 2014). In this study, we define a new role for arginine methylation in the regulation of R-loops. We show that the methylation of the RNA helicase DDX5 by PRMT5 regulates its association with XRN2 to suppress R-loops at transcription termination regions and maintain genomic stability. Results DDX5 resolves R-loops in vitro and in vivo DDX5 is an RGG/RG motif-containing helicase previously shown to unwind RNA:RNA and RNA:DNA duplexes (Hirling et al, 1989; Rossler et al, 2001; Xing et al, 2017). Dbp2, the S. cerevisiae homolog of DDX5, was shown to resolve RNA:DNA hybrids in the context of R-loops (Cloutier et al, 2016); however, whether DDX5 shares this R-loop resolving activity is unknown. To examine whether DDX5 resolves R-loops, we purified recombinant DDX5 to homogeneity (Fig 1A) and performed in vitro R-loop (RNA:DNA hybrid) and D-loop (DNA:DNA hybrid) unwinding activity assays using radiolabeled nucleotide substrates. The addition of increasing concentrations of DDX5 led to the appearance of a faster migrating species on native gels representing the DNA strands without the bound RNA fragment, and this occurred in an ATP-dependent manner (Fig 1B). Remarkably, DDX5 did not resolve the D-loop substrate (Fig 1B). These observations show that DDX5, like its yeast homolog Dbp2, resolves R-loops in vitro. Figure 1. DDX5 unwinds R-loops in vitro and represses cellular R-loop accumulation Coomassie Blue staining of recombinant human DDX5 purified in bacteria. M denotes the molecular mass markers in kDa. R-loop unwinding assay in the presence of increasing DDX5. The top panel shows a typical image obtained after the assay. The bar graph (bottom) shows the quantification. The average is expressed as percentage unwinding and standard error of the mean (SEM), n = 4. S9.6 signal in the nucleoplasm of U2OS cells. An average from three independent experiments performed in triplicate. In total, ˜90 images for each condition (10 images/slide and nine slides for each condition) were taken and two cells per image were quantified. Statistical significance was assessed using one-way ANOVA t-test. *P < 0.05; ****P < 0.0001. U2OS cells transfected with siCTL or siDDX5 were subjected to DRIP-qPCR analysis with anti-IgG and anti-S9.6 antibodies with or without RNase H treatment. The gene location and genomic qPCR amplification region are shown at the top of each panel. B, E, H, S, and X denote the location of the BsrGI, EcoRI, HindIII, SspI, and XbaI. The identified R-loop peaks were extracted from the R-loop database (R-loop DB) for each region. The bar graphs are the average and SEM from three independent experiments. Statistical significance was assessed using t-test. *P < 0.05. Source data are available online for this figure. Source Data for Figure 1 [embj2018100986-sup-0005-SDataFig1.pdf] Download figure Download PowerPoint To examine whether DDX5 resolves R-loops in vivo, we generated DDX5-deficient U2OS cells using three siRNAs and we measured the accumulation of RNA:DNA hybrids by immunofluorescence using the monoclonal antibody (S9.6) known to recognize RNA:DNA hybrids within the mitochondria, nucleoli, and the nucleoplasm (Ginno et al, 2012; Bhatia et al, 2014; Sollier et al, 2014; Hamperl et al, 2017; Hodroj et al, 2017). We measured the S9.6 signal in the nucleoplasm as the total nuclear signal subtracting the nucleolar contribution. The nucleus and nucleolus were detected with DAPI and anti-nucleolin antibodies, respectively (Appendix Fig S1A). Depletion of DDX5 led to a significant increase of the S9.6 signal in the nucleoplasm of U2OS cells with all siRNAs tested (Fig 1C). We also visualized the accumulation of RNA:DNA hybrids from isolated genomic DNA using slot-blot analysis with the S9.6 antibody in the presence or absence of RNase H. Again, we observed a significant increase in the S9.6 signal within the genomic DNA isolated from DDX5-deficient cells compared to control siRNA-treated cells. siSenataxin (siSTEX) was used as a positive control (Appendix Fig S1B and C). To examine the role of the DDX5 in R-loop resolution at precise genomic loci, we used the RNA:DNA immunoprecipitation (DRIP) method. By qPCR analysis, we quantified R-loop accumulation at four selected loci, EGR1, MALAT1, HIST1H2BG, and RPPH1, previously known to have a propensity to form R-loops (Yang et al, 2014; García-Rubio et al, 2015; Hodroj et al, 2017). The R-loops for EGR1 and MALAT1 reside within the gene body, while the R-loops for HIST1H2BG and RPPH1 encompass the transcription termination region, as determined previously and presented in the R-loop database (Fig 1D) (Wongsurawat et al, 2012). The knockdown of DDX5 resulted in a significant increase in R-loops at all four loci when compared to control siRNA conditions, and these were sensitive to RNase H treatment (Fig 1D). These findings suggest that DDX5-deficient cells accumulate R-loops. DDX5 deficiency leads to spontaneous DNA damage and hypersensitivity of U2OS cells to replication stress As unresolved R-loops result in DNA damage (Skourti-Stathaki et al, 2011), we next investigated whether DDX5-deficient cells exhibit spontaneous DNA damage due to unresolved R-loops, and indeed, this was the case, as assessed by 1) an increase in the phosphorylation of H2AX termed γH2AX by immunoblotting (Appendix Fig S2A) and an increase in the number of γH2AX foci per nuclei (Appendix Fig S2B). Cells deficient in DDX5 are known to be sensitive to ionizing radiation (Nicol et al, 2013); however, it is unknown whether these cells are sensitive to DNA replication stress such as hydroxyurea, which lead to R-loop accumulation during DNA replication (Hamperl et al, 2017). Depletion of DDX5 using two different siRNAs led to a significant reduction of cell survival to hydroxyurea treatment compared with the control siRNA-transfected cells (Appendix Fig S2C). To further confirm this effect, we also performed a FACS (fluorescence-activated cell sorting)-based survival analysis of co-cultured cells (outlined in Appendix Fig S2D). This analysis enables direct comparison in the same culture of the proliferative fitness of DDX5-expressing and depleted cells. The U2OS cells without or with stable GFP expression were transfected with control and DDX5 siRNAs, respectively. The cells were mixed and co-plated 2 days after transfection and then treated with hydroxyurea. Compared to non-treated cells, a decrease in the percentage of GFP-positive cells after treatment indicates an increase in sensitivity to hydroxyurea in the target siDDX5-transfected cells (Appendix Fig S2E). Depletion of DDX5 caused significant increase in cell sensitivity to hydroxyurea, either at low dose (0.4 mM) with long-time incubation (24 h) or at high dose (10 mM) and short exposure (2 h; Appendix Fig S2F and G). Taken together, these results suggest that DDX5 deficiency causes cell hypersensitivity to the DNA replication stress agent hydroxyurea, a known R-loop inducer (Hamperl et al, 2017). DDX5 is arginine-methylated in U2OS cells Interestingly, in a separate study, we identified DDX5 by mass spectrometry analysis as a PRMT5-interacting protein in two prostate cancer cell lines (Dataset EV1). DDX5 bears an arginine-rich motif, raising the possibility that DDX5 functions in R-loop homeostasis are regulated by arginine methylation. Indeed, PRMT5 is known to catalyze the mono- and symmetrical dimethylation of arginine residues in proteins (Branscombe et al, 2001). We first confirmed the physical association between PRMT5 and DDX5 in U2OS cells. Cells were lysed, and either PRMT5 or DDX5 was immunoprecipitated followed by immunoblotting. Significant amounts of DDX5 were co-immunoprecipitated with anti-PRMT5 antibodies, but not control IgG (Fig 2A). The reverse was also observed, PRMT5 was co-immunoprecipitated with anti-DDX5 antibodies (Fig 2B). These findings confirm the association of PRMT5 with DDX5. Figure 2. DDX5 is arginine-methylated in U2OS cells, and PRMT5 deficiency causes cellular R-loops to accumulate A. U2OS cells were lysed, and the lysates were incubated with anti-PRMT5 antibody or immunoglobulin G (IgG) as control followed by protein A-Sepharose bead addition. The immunoprecipitated (IP) proteins or whole cell extracts (WCEs) were separated by SDS–PAGE and Western-blotted (WB) with the indicated antibodies. B. Same as panel (A) except anti-DDX5 antibody was used instead of anti-PRMT5 antibody for immunoprecipitation. C. The mass spectrum of R502 monomethylation from Flag-DDX5 expressed in U2OS. D. HEK293 cells transfected with Flag-DDX5 were lysed and immunoprecipitated with anti-Flag antibodies. The bound proteins were along with WCE from untransfected (−) and Flag-DDX5 (+)-transfected cells and were Western-blotted with anti-Flag and anti-methylarginine antibodies (MeR). E, F. The knockdown using siRNAs of the indicated gene in U2OS cells was confirmed by Western blotting, and duplicate cell cultures were analyzed by immunofluorescence with S9.6 and anti-nucleolin antibodies. A typical image is shown. The scale bar represents 10 μm. Graphs show the average of 3 independent experiments performed in triplicates. Statistical significance was assessed using one-way ANOVA t-test. **P < 0.01 and ****P < 0.0001; n.s.: not significant. Source data are available online for this figure. Source Data for Figure 2 [embj2018100986-sup-0006-SDataFig2.pdf] Download figure Download PowerPoint Next, to determine whether DDX5 is arginine-methylated, we first immunoprecipitated DDX5 from U2OS cells and analyzed it in the presence of methylarginines by mass spectrometry. We identified R502 within the C-terminal RGG/RG motif of DDX5 to harbor a monomethyl (Fig 2C). U2OS cells transfected with Flag-epitope-tagged DDX5 were immunoprecipitated with anti-Flag antibodies, and the bound proteins immunoblotted with anti-methylarginine antibodies. We detected that Flag-DDX5 was arginine-methylated (Fig 2D). Deficiency of PRMT5 leads to increase in cellular R-loop accumulation RGG/RG motifs are methylated mainly by PRMT1 and PRMT5, the two major enzymes responsible for cellular protein arginine methylation (Blanc & Richard, 2017). To identify which PRMT leads to R-loop accumulation, we turned to R-loop visualization using immunofluorescence assays with S9.6 antibody using siPRMT1 or siPRMT5 U2OS cells (Fig 2E). We observed that the knockdown of PRMT5 such as DDX5, but not PRMT1, led to significant increase of nuclear S9.6 staining in U2OS cells (Fig 2F). Furthermore, PRMT5-depleted cells had increased spontaneous DNA damage, as assessed by γH2AX intensity (Appendix Fig S3), consistent with the appearance of unsolved R-loops. To further confirm that DDX5 and PRMT5 deficiencies cause R-loop accumulation, we used the DNA damage at RNA transcription (DART) system, which measures an RNA:DNA hybrid at a particular locus (Teng et al, 2018; Liang et al, 2019). The light-inducible chromophore-modified KillerRed (KR) is fused with either transcription activator (TA) or repressor (tetR). KR generates reactive oxygen species (ROS) through the excited chromophore and induces DNA damage and transcriptional activation at the genome-integrated tet response element (TRE) locus in the U2OS TRE cells. Elevated R-loop at the TRE locus over background is visualized by S9.6 immunofluorescence (Teng et al, 2018; Liang et al, 2019). DDX5 or PRMT5 knockdown (Fig 3A) led to a significant increase in R-loop specifically at the TA-KR marked locus, while the level of R-loops was similar to the control at the TetR-KR locus (Fig 3B and C). The signal we observe is similar to published studies (Teng et al, 2018). Under the conditions used (the staining involves a heating step on a 95°C heating block for 20 min to expose the antigen and blocking with 5% BSA), the S9.6 focus is more apparent than the typical nucleolar staining observed with standard S9.6 staining protocols. The fact that the cellular system is using a defined locus where multiple breaks are induced by KillerRed, this induces transcription very efficiently leading to R-loop accumulation and a stronger signal over typical nucleolar staining. These finding further confirm the accumulation of R-loops in the absence of DDX5 and PRMT5 using an independent assay, i.e., DART. Figure 3. Depletion of DDX5 increases R-loops at transcribed regions with local ROS-induced DNA damage Western blotting of PRMT5 and DDX5 knockdowns. Representative images of S9.6 staining in siCTL, siDDX5, or siPRMT5 knockdown at transcription on (TA-KR) or off (TetR-KR) genomic loci in U2OS TRE cells. The nuclei are visualized by DAPI. Quantification of the S9.6 foci intensity in the indicated conditions. Three independent experiments were performed, and 50 cells were analyzed in each experiment. Error bars represent mean of S9.6 foci intensity quantification with SEM. The statistical analysis was performed by two-tailed Student's t-test (Mann–Whitney U-test). ****P < 0.0001. Source data are available online for this figure. Source Data for Figure 3 [embj2018100986-sup-0007-SDataFig3.pdf] Download figure Download PowerPoint We next performed DRIP-qPCR analysis with siPRMT5-transfected cells using the same four loci that showed accumulation of R-loops using siDDX5 (Fig 1D). PRMT5-depleted U2OS cells also displayed an increase in R-loops in an RNase H-dependent manner at the EGR1, MALAT1, HIST1H2BG, and RPPH1 loci (Fig 4A). To further show that DDX5 and PRMT5 are linked in the same pathway, we performed a double knockdown (Fig 4B) and assessed R-loops at the same loci. The double depletion did not reveal a synergistic increase in R-loop accumulation than the single depletion of either PRMT5 or DDX5 (Fig 4C), suggesting PRMT5 and DDX5 are functionally linked for this function. Figure 4. PRMT5-deficient cells accumulate R-loops at specific loci U2OS cells transfected with siCTL or siPRMT5 were subjected to DRIP-qPCR analysis. The average and SEM from three independent experiments are shown. Statistical significance was assessed using Student's t-test. *P < 0.05 and **P < 0.01. Western blotting of whole cell extracts from siCTL-, siPRMT5-, siDDX5-, or siDDX5/ siPRMT5-transfected cells. U2OS cells transfected with siCTL, siPRMT5, siDDX5 or siDDX5/ siPRMT5 were subjected to DRIP-qPCR analysis. The average and SEM from three independent experiments are shown. Statistical significance was assessed using Student's t-test. *P < 0.05 and **P < 0.01. Source data are available online for this figure. Source Data for Figure 4 [embj2018100986-sup-0008-SDataFig4.pdf] Download figure Download PowerPoint DDX5 is methylated by PRMT5 at its C-terminal RGG motif Flag-DDX5 was expressed in U2OS cells, and anti-Flag immunoprecipitations were separated by SDS–PAGE and the bound proteins immunoblotted with anti-symmetrical dimethylarginine antibodies (SMDAs). We observed that PRMT5 knockdown caused a significant reduction in the symmetrical arginine dimethylation of DDX5 (Fig 5A). These findings confirm that DDX5 is indeed an in vivo substrate of PRMT5. To map the methylated region, we next performed in vitro arginine methylation analysis using glutathione-S-transferase (GST)–DDX5 fusion proteins. DDX5 has two RGG/RG motifs: One located at its N-terminus and another at its C-terminus. Interestingly, both DDX5 RGG/RG motifs are conserved in the yeast homolog Dbp2 (Fig 5B). We generated three truncation mutants of DDX5, including the N-terminal region (residues 1–100; F1), the central catalytic enzyme domain (92–471; F2), and the C-terminal region (residues 466–614; F3; Fig 5C). Only the C-terminal region (F3), encompassing the RGG/RG motif, was methylated by PRMT5 (Fig 5D). We then substituted DDX5 R478, R482, R484, R486, and R502 within the RGG/RG motif with lysines in a smaller region (466–555; F4). The 5R to 5K mutation within the F4 fragment of DDX5 (RK) completely abolished arginine methylation by PRMT5 (Fig 5E). Figure 5. The RGG/RG motif of DDX5 is a substrate for PRMT5 and is required for R-loop resolution in vivo A. U2OS cells were transfected with a Flag-DDX5 expression vector in the presence of siCTL or siPRMT5. Lysates were immunoprecipitated with IgG or anti-Flag antibodies as indicated. The WCE and the bound proteins were Western-blotted with anti-symmetrical dimethylarginine antibody or anti-Flag antibodies. B. A schematic of DDX5 helicase domain and RGG/RG motifs is shown. C. GST fusion of DDX5 fragments (F1-F4) and the RK mutant used in this study. RK (*) indicates R to K substitutions of 5 arginines (R478, R482, R484, R486, and R502). D, E. Coomassie Blue (left panel) and in vitro methylation assay (right panel) of indicated GST-DDX5 fragments and the GST-DDX5-RK mutant. F. Immunofluorescence analysis with S9.6 and anti-Flag antibodies of U2OS cells transfected with Flag-tagged DDX5, DDX5-RK, or DDX5-XD (helicase dead). Nuclear S9.6 signal was counted in both Flag-negative and Flag-positive cells. The Flag-negative cells were considered as untransfected cells. The graphs shown represent the quantification with the SEM from three independent experiments. Statistical significance was assessed using one-way ANOVA t-test. ****P < 0.0001. G. Immunofluorescence analysis with S9.6 and anti-Flag antibodies of U2OS cells transfected with siCTL or siDDX5-1 and Flag-tagged DDX5 (+WT) or DDX5-RK (+RK) as indicated. The graphs show the average and SEM from three" @default.
• W2952073075 created "2019-06-27" @default.
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• W2952073075 title "Arginine methylation of the <scp>DDX</scp> 5 helicase <scp>RGG</scp> / <scp>RG</scp> motif by <scp>PRMT</scp> 5 regulates resolution of RNA:DNA hybrids" @default.
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