DDX47 untangles R-loops with only certain other helicases.

R-loops, formed transiently during gene transcription, are tightly controlled to avoid conflict with ongoing processes. Marchena-Cruz et al. identified DExD/H box RNA helicase DDX47 using a new R-loop resolving screen and defined a unique role for this helicase in nucleolar R-loops and its interplay with senataxin (SETX) and DDX39B.

PRMT7 ablation stimulates anti-tumor immunity and sensitizes melanoma to immune checkpoint blockade.

Despite the success of immune checkpoint inhibitor (ICI) therapy for cancer, resistance and relapse are frequent. Combination therapies are expected to enhance response rates and overcome this resistance. Herein, we report that combining PRMT7 inhibition with ICI therapy induces a strong anti-tumor T cell immunity and restrains tumor growth in vivo by increasing immune cell infiltration. PRMT7-deficient B16.F10 melanoma exhibits increased expression of genes in the interferon pathway, antigen presentation, and chemokine signaling. PRMT7 deficiency or inhibition with SGC3027 in B16.F10 melanoma results in reduced DNMT expression, loss of DNA methylation in the regulatory regions of endogenous retroviral elements (ERVs) causing their increased expression. PRMT7-deficient cells increase RIG-I and MDA5 expression with a reduction in the H4R3me2s repressive histone mark at their gene promoters. Our findings identify PRMT7 as a regulatory checkpoint for RIG-I, MDA5, and their ERV-double-stranded RNA (dsRNA) ligands, facilitating immune escape and anti-tumor T cell immunity to restrain tumor growth.

Deletion of RBMX RGG/RG motif in Shashi-XLID syndrome leads to aberrant p53 activation and neuronal differentiation defects.

RNA-binding proteins play important roles in X-linked intellectual disability (XLID). In this study, we investigate the contribution of the XLID-associated RBMX in neuronal differentiation. We show that RBMX-depleted cells exhibit aberrant activation of the p53 pathway. Moreover, we identify that the RBMX RGG/RG motif is methylated by protein arginine methyltransferase 5 (PRMT5), and this regulates assembly with the SRSF1 splicing factor into higher-order complexes. Depletion of RBMX or disruption of the RBMX/SRSF1 complex in PRMT5-depleted cells reduces SRSF1 binding to the MDM4 precursor (pre-)mRNA, leading to exon 6 exclusion and lower MDM4 protein levels. Transcriptomic analysis of isogenic Shashi-XLID human-induced pluripotent stem cells (hiPSCs) generated using CRISPR-Cas9 reveals a dysregulation of MDM4 splicing and aberrant p53 upregulation. Shashi-XLID neural progenitor cells (NPCs) display differentiation and morphological abnormalities accompanied with excessive apoptosis. Our findings identify RBMX as a regulator of SRSF1 and the p53 pathway, suggesting that the loss of function of the RBMX RGG/RG motif is the cause of Shashi-XLID syndrome.

Arginine methylation of SARS-Cov-2 nucleocapsid protein regulates RNA binding, its ability to suppress stress granule formation, and viral replication.

Viral proteins are known to be methylated by host protein arginine methyltransferases (PRMTs) necessary for the viral life cycle, but it remains unknown whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins are methylated. Herein, we show that PRMT1 methylates SARS-CoV-2 nucleocapsid (N) protein at residues R95 and R177 within RGG/RG motifs, preferred PRMT target sequences. We confirmed arginine methylation of N protein by immunoblotting viral proteins extracted from SARS-CoV-2 virions isolated from cell culture. Type I PRMT inhibitor (MS023) or substitution of R95 or R177 with lysine inhibited interaction of N protein with the 5'-UTR of SARS-CoV-2 genomic RNA, a property required for viral packaging. We also defined the N protein interactome in HEK293 cells, which identified PRMT1 and many of its RGG/RG substrates, including the known interacting protein G3BP1 as well as other components of stress granules (SGs), which are part of the host antiviral response. Methylation of R95 regulated the ability of N protein to suppress the formation of SGs, as R95K substitution or MS023 treatment blocked N-mediated suppression of SGs. Also, the coexpression of methylarginine reader Tudor domain-containing protein 3 quenched N protein-mediated suppression of SGs in a dose-dependent manner. Finally, pretreatment of VeroE6 cells with MS023 significantly reduced SARS-CoV-2 replication. Because type I PRMT inhibitors are already undergoing clinical trials for cancer treatment, inhibiting arginine methylation to target the later stages of the viral life cycle such as viral genome packaging and assembly of virions may represent an additional therapeutic application of these drugs.

Synergistic effects of type I PRMT and PARP inhibitors against non-small cell lung cancer cells.

BACKGROUND: Non-small cell lung carcinoma (NSCLC) is a leading cause of cancer-related death and represents a major health burden worldwide. Current therapies for NSCLC include chemotherapy, immunotherapy, and targeted molecular agents such as tyrosine kinase inhibitors and epigenetic drugs such as DNA methyltransferase inhibitors. However, survival rates remain low for patients with NSCLC, especially those with metastatic disease. A major cause for therapeutic failure is drug resistance, highlighting the need for novel therapies and combination strategies. Given that epigenetic modulators such as protein arginine methyltransferases (PRMTs) are frequently overexpressed in cancers, PRMT inhibitors are a promising class of cancer therapeutics. We screened a library of epigenetic and anticancer drugs to identify compounds that would synergize with MS023, a type I PRMT inhibitor, in decreasing the viability of methylthioadenosine phosphorylase (MTAP)-negative NSCLC cells. RESULTS: Among 181 compounds, we identified PARP inhibitors (PARPi) as having a strong synergistic interaction with type I PRMT inhibition. The combination of MS023 and the PARP inhibitor BMN-673 (Talazoparib) demonstrated strong synergistic interaction at low nanomolar concentrations in MTAP-negative NSCLC cell lines A549, SK-LU-1 and HCC4006. The re-introduction of MTAP decreased the sensitivity of the combination therapy in A549. The combination therapy resulted in elevated γ-H2AX foci indicating increased DNA damage causing decreased cell viability. Lastly, the combination therapy was effective in PARPi resistant ovarian cancer cells, suggesting that type I PRMT inhibitors could mitigate PARPi resistance, thus potentially having an important clinical impact for cancer treatment. CONCLUSIONS: These findings identify a novel cancer drug combination therapy, which is more potent than the separate single-agent therapies. Thus, combining PARP inhibitors and type I PRMT inhibitors represents a new therapeutic opportunity for MTAP-negative NSCLC and certain cancer cells resistant to PARP inhibitors.

DDX5 resolves R-loops at DNA double-strand breaks to promote DNA repair and avoid chromosomal deletions.

R-loops are three-stranded structures consisting of a DNA/RNA hybrid and a displaced DNA strand. The regulatory factors required to process this fundamental genetic structure near double-strand DNA breaks (DSBs) are not well understood. We previously reported that cellular depletion of the ATP-dependent DEAD box RNA helicase DDX5 increases R-loops genome-wide causing genomic instability. In this study, we define a pivotal role for DDX5 in clearing R-loops at or near DSBs enabling proper DNA repair to avoid aberrations such as chromosomal deletions. Remarkably, using the non-homologous end joining reporter gene (EJ5-GFP), we show that DDX5-deficient U2OS cells exhibited asymmetric end deletions on the side of the DSBs where there is overlap with a transcribed gene. Cross-linking and immunoprecipitation showed that DDX5 bound RNA transcripts near DSBs and required its helicase domain and the presence of DDX5 near DSBs was also shown by chromatin immunoprecipitation. DDX5 was excluded from DSBs in a transcription- and ATM activation-dependent manner. Using DNA/RNA immunoprecipitation, we show DDX5-deficient cells had increased R-loops near DSBs. Finally, DDX5 deficiency led to delayed exonuclease 1 and replication protein A recruitment to laser irradiation-induced DNA damage sites, resulting in homologous recombination repair defects. Our findings define a role for DDX5 in facilitating the clearance of RNA transcripts overlapping DSBs to ensure proper DNA repair.

Genome-wide R-loop analysis defines unique roles for DDX5, XRN2, and PRMT5 in DNA/RNA hybrid resolution.

DDX5, XRN2, and PRMT5 have been shown to resolve DNA/RNA hybrids (R-loops) at RNA polymerase II transcription termination sites at few genomic loci. Herein, we perform genome-wide R-loop mapping using classical DNA/RNA immunoprecipitation and high-throughput sequencing (DRIP-seq) of loci regulated by DDX5, XRN2, and PRMT5. We observed hundreds to thousands of R-loop gains and losses at transcribed loci in DDX5-, XRN2-, and PRMT5-deficient U2OS cells. R-loop gains were characteristic of highly transcribed genes located at gene-rich regions, whereas R-loop losses were observed in low-density gene areas. DDX5, XRN2, and PRMT5 shared many R-loop gain loci at transcription termination sites, consistent with their coordinated role in RNA polymerase II transcription termination. DDX5-depleted cells had unique R-loop gain peaks near the transcription start site that did not overlap with those of siXRN2 and siPRMT5 cells, suggesting a role for DDX5 in transcription initiation independent of XRN2 and PRMT5. Moreover, we observed that the accumulated R-loops at certain loci in siDDX5, siXRN2, and siPRMT5 cells near the transcription start site of genes led to antisense intergenic transcription. Our findings define unique and shared roles of DDX5, XRN2, and PRMT5 in DNA/RNA hybrid regulation.

Arginine methylation of the DDX5 helicase RGG/RG motif by PRMT5 regulates resolution of RNA:DNA hybrids.

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.

Loss of PRMT5 Promotes PDGFRα Degradation during Oligodendrocyte Differentiation and Myelination.

The oligodendrocyte lineage is responsible for myelination of the central nervous system. Post-translational modifications are known to regulate oligodendrocyte precursor cell (OPC) differentiation into mature myelinating oligodendrocytes. The role of arginine methylation during oligodendrocyte differentiation and myelination is still poorly understood. We generated mice depleted of PRMT5 in OPCs using Olig2-Cre, and these mice developed severe hypomyelination and died at the third post-natal week. PRMT5-deficient cells have lower levels of PDGFRα at the plasma membrane due to increased degradation by the Cbl E3 ligase. Mechanistically, the loss of arginine methylation at R554 of the PDGFRα intracellular domain unmasks a Cbl binding site at Y555. We observed the progressive decrease in PRMT5 during oligodendrocyte differentiation, and we show that one role of this decrease is to downregulate growth signals provided by PDGFRα to initiate oligodendrocyte differentiation and myelination. More broadly, the inhibition of PRMT5 may be used therapeutically to manipulate PDGFRα bioavailability.

GFI1 facilitates efficient DNA repair by regulating PRMT1 dependent methylation of MRE11 and 53BP1.

GFI1 is a transcriptional regulator expressed in lymphoid cells, and an "oncorequisite" factor required for development and maintenance of T-lymphoid leukemia. GFI1 deletion causes hypersensitivity to ionizing radiation, for which the molecular mechanism remains unknown. Here, we demonstrate that GFI1 is required in T cells for the regulation of key DNA damage signaling and repair proteins. Specifically, GFI1 interacts with the arginine methyltransferase PRMT1 and its substrates MRE11 and 53BP1. We demonstrate that GFI1 enables PRMT1 to bind and methylate MRE11 and 53BP1, which is necessary for their function in the DNA damage response. Thus, our results provide evidence that GFI1 can adopt non-transcriptional roles, mediating the post-translational modification of proteins involved in DNA repair. These findings have direct implications for treatment responses in tumors overexpressing GFI1 and suggest that GFI1's activity may be a therapeutic target in these malignancies.

Lysine methylation of FEN1 by SET7 is essential for its cellular response to replicative stress.

The DNA damage response (DDR) is central to the cell survival and it requires post-translational modifications, in part, to sense the damage, amplify the signaling response and recruit and regulate DNA repair enzymes. Lysine methylation of histones such as H4K20 and non-histone proteins including p53 has been shown to be essential for the mounting of the DDR. It is well-known that the lysine methyltransferase SET7 regulates the DDR, as cells lacking this enzyme are hypersensitive to chemotherapeutic drugs. To define addition substrates of SET7 involved in the DDR, we screened a peptide array encompassing potential lysine methylation sites from >100 key DDR proteins and identified peptides from 58 proteins to be lysine methylated defining a methylation consensus sequence of [S>K-2; S>R-1; K0] consistent with previous findings. We focused on K377 methylation of the Flap endonuclease 1 (FEN1), a structure specific endonuclease with important functions in Okazaki fragment processing during DNA replication as a substrate of SET7. FEN1 was monomethylated by SET7 in vivo in a cell cycle dependent manner with levels increasing as cells progressed through S phase and decreasing as they exited S phase, as detected using K377me1 specific antibodies. Although K377me1 did not affect the enzymatic activity of FEN1, it was required for the cellular response to replicative stress by FEN1. These finding define FEN1 as a new substrate of SET7 required for the DDR.

CTCF facilitates DNA double-strand break repair by enhancing homologous recombination repair.

The repair of DNA double-strand breaks (DSBs) is mediated via two major pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR) repair. DSB repair is vital for cell survival, genome stability, and tumor suppression. In contrast to NHEJ, HR relies on extensive homology and templated DNA synthesis to restore the sequence surrounding the break site. We report a new role for the multifunctional protein CCCTC-binding factor (CTCF) in facilitating HR-mediated DSB repair. CTCF is recruited to DSB through its zinc finger domain independently of poly(ADP-ribose) polymers, known as PARylation, catalyzed by poly(ADP-ribose) polymerase 1 (PARP-1). CTCF ensures proper DSB repair kinetics in response to γ-irradiation, and the loss of CTCF compromises HR-mediated repair. Consistent with its role in HR, loss of CTCF results in hypersensitivity to DNA damage, inducing agents and inhibitors of PARP. Mechanistically, CTCF acts downstream of BRCA1 in the HR pathway and associates with BRCA2 in a PARylation-dependent manner, enhancing BRCA2 recruitment to DSB. In contrast, CTCF does not influence the recruitment of the NHEJ protein 53BP1 or LIGIV to DSB. Together, our findings establish for the first time that CTCF is an important regulator of the HR pathway.

Arginine Methylation by PRMT1 Regulates Muscle Stem Cell Fate.

Quiescent muscle stem cells (MSCs) become activated in response to skeletal muscle injury to initiate regeneration. Activated MSCs proliferate and differentiate to repair damaged fibers or self-renew to maintain the pool and ensure future regeneration. The balance between self-renewal, proliferation, and differentiation is a tightly regulated process controlled by a genetic cascade involving determinant transcription factors such as Pax7, Myf5, MyoD, and MyoG. Recently, there have been several reports about the role of arginine methylation as a requirement for epigenetically mediated control of muscle regeneration. Here we report that the protein arginine methyltransferase 1 (PRMT1) is expressed in MSCs and that conditional ablation of PRMT1 in MSCs using Pax7CreERT2 causes impairment of muscle regeneration. Importantly, PRMT1-deficient MSCs have enhanced cell proliferation after injury but are unable to terminate the myogenic differentiation program, leading to regeneration failure. We identify the coactivator of Six1, Eya1, as a substrate of PRMT1. We show that PRMT1 methylates Eya1 in vitro and that loss of PRMT1 function in vivo prevents Eya1 methylation. Moreover, we observe that PRMT1-deficient MSCs have reduced expression of Eya1/Six1 target MyoD due to disruption of Eya1 recruitment at the MyoD promoter and subsequent Eya1-mediated coactivation. These findings suggest that arginine methylation by PRMT1 regulates muscle stem cell fate through the Eya1/Six1/MyoD axis.

Aven recognition of RNA G-quadruplexes regulates translation of the mixed lineage leukemia protooncogenes.

G-quadruplexes (G4) are extremely stable secondary structures forming stacks of guanine tetrads. DNA G4 structures have been extensively studied, however, less is known about G4 motifs in mRNAs, especially in their coding sequences. Herein, we show that Aven stimulates the mRNA translation of the mixed lineage leukemia (MLL) proto-oncogene in an arginine methylation-dependent manner. The Aven RGG/RG motif bound G4 structures within the coding regions of the MLL1 and MLL4 mRNAs increasing their polysomal association and translation, resulting in the induction of transcription of leukemic genes. The DHX36 RNA helicase associated with the Aven complex and was required for optimal translation of G4 mRNAs. Depletion of Aven led to a decrease in synthesis of MLL1 and MLL4 proteins resulting in reduced proliferation of leukemic cells. These findings identify an Aven-centered complex that stimulates the translation of G4 harboring mRNAs, thereby promoting survival of leukemic cells.

Arginine methylation of hnRNPUL1 regulates interaction with NBS1 and recruitment to sites of DNA damage.

Arginine methylation is a post-translational modification required for the maintenance of genomic integrity. Cells deficient in protein arginine methyltransferase 1 (PRMT1) have DNA damage signaling defects, defective checkpoint activation and extensive genomic instability. Herein we identify the DNA damage protein and RNA binding protein, hnRNPUL1, to be a substrate of PRMT1. We identify the dimethylation of R584, R618, R620, R645, and R656, as well as the monomethylation of R661 R685 and R690 within hnRNPUL1 in U2OS cells by mass spectrometry. Moreover, we define the arginines within the RGG/RG motifs as the site of methylation by PRMT1 both in vitro and in vivo. The arginines 612, 618, 620, 639, 645, 656 and 661 within the human hnRNPUL1 RGG/RG motifs were substituted with lysines to generate hnRNPUL1(RK). hnRNPUL1(RK) was hypomethylated and lacked the ability to interact with PRMT1, unlike wild type hnRNPUL1. Co-immunoprecipitation studies showed that hnRNPUL1(RK) had impaired ability to associate with the DNA damage protein NBS1. Moreover, hnRNPUL1(RK) was not recruited to sites of DNA damage, unlike wild type hnRNPUL1, in the presence of transcriptional inhibitors. These findings define a role for arginine methylation during the DNA damage response to regulate protein-protein interactions for the recruitment at sites of damage.

Evaluation of homogeneity and genetic stability of REOLYSIN (pelareorep) by complete genome sequencing of reovirus after large scale production.

REOLYSIN (pelareorep) is a proprietary isolate of the reovirus T3D (Type 3 Dearing) strain which is currently being tested in clinical trials as an anticancer therapeutic agent. Reovirus genomes are composed of ten segments of double-stranded ribonucleic acid (RNA) characterized by genome size: large (L1, L2, and L3), medium (M1, M2, and M3), and small (S1, S2, S3, and S4). The objective of this work was to evaluate the homogeneity and genetic stability of REOLYSIN. Sanger sequencing (SS) performed on test articles derived from the Master Virus Bank (MVB) and Working Virus Bank (WVB) identified many modifications when compared to GenBank reference sequences. Massively parallel sequencing (MPS) using Roche-454 sequencing was performed on REOLYSIN (100 L scale) and resulted in 69,821,115 bases and an average of 335 bases per read. Twenty-nine high confidence differences relative to the GenBank reference sequence were identified in REOLYSIN by MPS. Of those, 27 were previously identified by SS in the virus bank-derived test articles. Of the remaining two nucleotide differences, one was predicted to be silent at the amino acid level (L3 genome-T3163C, codon 1054, 86% of the population was "T" and 13% of the population were reported as "C"). The other modification was in the noncoding region (M1 genome-A2284A to A2284G), and A2284G was present in 97% of the population. The results obtained from MPS were comparable to those from SS; both demonstrate a high level of homogeneity at the amino acid level and genetic stability of REOLYSIN. Finally, phylogenetic analysis of the REOLYSIN L1 genome segment showed close evolutionary relationship with its human homologs, serotypes Lang and Dearing.

The QKI-5 and QKI-6 RNA binding proteins regulate the expression of microRNA 7 in glial cells.

The quaking (qkI) gene encodes 3 major alternatively spliced isoforms that contain unique sequences at their C termini dictating their cellular localization. QKI-5 is predominantly nuclear, whereas QKI-6 is distributed throughout the cell and QKI-7 is cytoplasmic. The QKI isoforms are sequence-specific RNA binding proteins expressed mainly in glial cells modulating RNA splicing, export, and stability. Herein, we identify a new role for the QKI proteins in the regulation of microRNA (miRNA) processing. We observed that small interfering RNA (siRNA)-mediated QKI depletion of U343 glioblastoma cells leads to a robust increase in miR-7 expression. The processing from primary to mature miR-7 was inhibited in the presence QKI-5 and QKI-6 but not QKI-7, suggesting that the nuclear localization plays an important role in the regulation of miR-7 expression. The primary miR-7-1 was bound by the QKI isoforms in a QKI response element (QRE)-specific manner. We observed that the pri-miR-7-1 RNA was tightly bound to Drosha in the presence of the QKI isoforms, and this association was not observed in siRNA-mediated QKI or Drosha-depleted U343 glioblastoma cells. Moreover, the presence of the QKI isoforms led to an increase presence of pri-miR-7 in nuclear foci, suggesting that pri-miR-7-1 is retained in the nucleus by the QKI isoforms. miR-7 is known to target the epidermal growth factor (EGF) receptor (EGFR) 3' untranslated region (3'-UTR), and indeed, QKI-deficient U343 cells had reduced EGFR expression and decreased ERK activation in response to EGF. Elevated levels of miR-7 are associated with cell cycle arrest, and it was observed that QKI-deficient U343 that harbor elevated levels of miR-7 exhibited defects in cell proliferation that were partially rescued by the addition of a miR-7 inhibitor. These findings suggest that the QKI isoforms regulate glial cell function and proliferation by regulating the processing of certain miRNAs.

Arginine methylation by PRMT1 regulates nuclear-cytoplasmic localization and toxicity of FUS/TLS harbouring ALS-linked mutations.

Mutations in FUS/TLS (fused in sarcoma/translated in liposarcoma) cause an inheritable form of amyotrophic lateral sclerosis (ALS6). In contrast to FUS(WT), which is concentrated in the nucleus, these mutants are abnormally distributed in the cytoplasm where they form inclusions and associate with stress granules. The data reported herein demonstrate the importance of protein arginine methylation in nuclear-cytoplasmic shuttling of FUS and abnormalities of ALS-causing mutants. Depletion of protein arginine methyltransferase 1 (PRMT1; the enzyme that methylates FUS) in mouse embryonic fibroblasts by gene knockout, or in human HEK293 cells by siRNA knockdown, diminished the ability of ALS-linked FUS mutants to localize to the cytoplasm and form inclusions. To examine properties of FUS mutants in the context of neurons vulnerable to the disease, FUS(WT) and ALS-linked FUS mutants were expressed in motor neurons of dissociated murine spinal cord cultures. In motor neurons, shRNA-mediated PRMT1 knockdown concomitant with the expression of FUS actually accentuated the shift in distribution of ALS-linked FUS mutants from the nucleus to the cytoplasm. However, when PRMT1 was inhibited prior to expression of ALS-linked FUS mutants, by pretreatment with a global methyltransferase inhibitor, ALS-linked FUS mutants were sequestered in the nucleus and cytoplasmic inclusions were reduced, as in the cell lines. Mitochondria were significantly shorter in neurons with cytoplasmic ALS-linked FUS mutants, a factor that could contribute to toxicity. We propose that arginine methylation by PRMT1 participates in the nuclear-cytoplasmic shuttling of FUS, particularly of ALS6-associated mutants, and thus contributes to the toxic gain of function conferred by these disease-causing mutations.

The MRE11 GAR motif regulates DNA double-strand break processing and ATR activation.

The MRE11/RAD50/NBS1 complex is the primary sensor rapidly recruited to DNA double-strand breaks (DSBs). MRE11 is known to be arginine methylated by PRMT1 within its glycine-arginine-rich (GAR) motif. In this study, we report a mouse knock-in allele of Mre11 that substitutes the arginines with lysines in the GAR motif and generates the MRE11(RK) protein devoid of methylated arginines. The Mre11(RK/RK) mice were hypersensitive to γ-irradiation (IR) and the cells from these mice displayed cell cycle checkpoint defects and chromosome instability. Moreover, the Mre11(RK/RK) MEFs exhibited ATR/CHK1 signaling defects and impairment in the recruitment of RPA and RAD51 to the damaged sites. The M(RK)RN complex formed and localized to the sites of DNA damage and normally activated the ATM pathway in response to IR. The M(RK)RN complex exhibited exonuclease and DNA-binding defects in vitro responsible for the impaired DNA end resection and ATR activation observed in vivo in response to IR. Our findings provide genetic evidence for the critical role of the MRE11 GAR motif in DSB repair, and demonstrate a mechanistic link between post-translational modifications at the MRE11 GAR motif and DSB processing, as well as the ATR/CHK1 checkpoint signaling.