A glycine-arginine domain in control of the human MRE11 DNA repair protein

Human MRE11 is a key enzyme in DNA double-strand break repair and genome stability. Human MRE11 bears a glycine-arginine-rich (GAR) motif that is conserved among multicellular eukaryotic species. We investigated how this motif influences MRE11 function. Human MRE11 alone or a complex of MRE11, RAD50, and NBS1 (MRN) was methylated in insect cells, suggesting that this modification is conserved during evolution. We demonstrate that PRMT1 interacts with MRE11 but not with the MRN complex, suggesting that MRE11 arginine methylation occurs prior to the binding of NBS1 and RAD50. Moreover, the first six methylated arginines are essential for the regulation of MRE11 DNA binding and nuclease activity. The inhibition of arginine methylation leads to a reduction in MRE11 and RAD51 focus formation on a unique double-strand break in vivo. Furthermore, the MRE11-methylated GAR domain is sufficient for its targeting to DNA damage foci and colocalization with gamma-H2AX. These studies highlight an important role for the GAR domain in regulating MRE11 function at the biochemical and cellular levels during DNA double-strand break repair.     

The GAR Motif of 53BP1 is Arginine Methylated by PRMT1 and is Necessary for 53BP1 DNA Binding Activity

The p53-binding protein 1 (53BP1) is rapidly recruited to sites of DNA double-strand breaks and forms characteristics nuclear foci, demonstrating its role in the early events of detection, signaling and repair of damaged DNA. 53BP1 contains a glycine arginine rich (GAR) motif of unknown function within its kinetochore binding domain. Herein, we show that the GAR motif of 53BP1 is arginine methylated by protein arginine methyltransferase 1 (PRMT1), the same methyltransferase that methylates MRE11. 53BP1 contains asymmetric dimethylarginines (aDMA) within cells, as detected with methylarginine-specific antibodies. Amino acid substitution of the arginines within the GAR motif of 53BP1 abrogated binding to single and double-stranded DNA, demonstrating that the GAR motif is required for DNA binding activity of 53BP1. Fibroblast cells treated with methylase inhibitors failed to relocalize 53BP1 to sites of DNA damage and formed few ?-H2AX foci, consistent with our previous data that MRE11 fails to relocalize to DNA damage sites in cells treated with methylase inhibitors. Our findings identify the GAR motif as a region required for 53BP1 DNA binding activity and is the site of methylation by PRMT1.     

Protein arginine methyltransferase 1 regulates herpes simplex virus replication through ICP27 RGG-box methylation

Protein arginine methylation is involved in viral infection and replication through the modulation of diverse cellular processes including RNA metabolism, cytokine signaling, and subcellular localization. It has been suggested previously that the protein arginine methylation of the RGG-box of ICP27 is required for herpes simplex virus type-1 (HSV-1) viral replication and gene expression in vivo. However, a cellular mediator for this process has not yet been identified. In our current study, we show that the protein arginine methyltransferase 1 (PRMT1) is a cellular mediator of the arginine methylation of ICP27 RGG-box. We generated arginine substitution mutants in this domain and examined which arginine residues are required for methylation by PRMT1. R138, R148 and R150 were found to be the major sites of this methylation but additional arginine residues serving as minor methylation sites are still required to sustain the fully methylated form of ICP27 RGG. We also demonstrate that the nuclear foci-like structure formation, SRPK interactions, and RNA-binding activity of ICP27 are modulated by the arginine methylation of the ICP27 RGG-box. Furthermore, HSV-1 replication is inhibited by hypomethylation of this domain resulting from the use of general PRMT inhibitors or arginine mutations. Our data thus suggest that the PRMT1 plays a key role as a cellular regulator of HSV-1 replication through ICP27 RGG-box methylation.     

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.     

Protein arginine methylation of SERBP1 by protein arginine methyltransferase 1 affects cytoplasmic/nuclear distribution

Protein arginine methylation regulates a broad array of cellular processes. SERBP1 implicated in tumor progression through its putative involvement in the plaminogen activator protease cascade, is an RNA-binding protein containing an RG-rich domain and an RGG box domain that might be methylated by protein arginine N-methyltransferases (PRMTs). Asymmetric dimethylarginine (aDMA) was detected in SERBP1 and an indirect methyltransferase inhibitor adenosine dialdehyde (AdOx) significantly reduced the methylation signals. Arginines in the middle RG and C-terminal RGG region of SERBP1 are methylated based on the analyses of different deletion constructs. The predominant type I protein arginine methyltransferase PRMT1 co-immunoprecipitated with SERBP1 and the level of bound PRMT1 decreased upon the addition of AdOx. Recombinant PRMT1 methylated SERBP1 and knockdown of PRMT1 significantly reduced the aDMA level of SERBP1, indicating that SERBP1 is specifically methylated by PRMT1. Immunofluorescent analyses of endogenous SERBP1 showed predominant cytoplasmic localization of SERBP1. Treatment of AdOx or PRMT1 siRNA increased the nuclear localization of SERBP1. Analyses of different deletions indicated that the middle RG region is important for the nuclear localization while both N- and C- terminus are required for nuclear export. Low methylation of the C-terminal RGG region also favors nuclear localization. In conclusion, the RG-rich and RGG box of SERBP1 is asymmetrically dimethylated by PRMT1 and the modification affects protein interaction and intracellular localization of the protein. These findings provide the basis for dissecting the roles of SERBP1.     

Sam68 RNA binding protein is an in vivo substrate for protein arginine N-methyltransferase 1

RNA binding proteins often contain multiple arginine glycine repeats, a sequence that is frequently methylated by protein arginine methyltransferases. The role of this posttranslational modification in the life cycle of RNA binding proteins is not well understood. Herein, we report that Sam68, a heteronuclear ribonucleoprotein K homology domain containing RNA binding protein, associates with and is methylated in vivo by the protein arginine N-methyltransferase 1 (PRMT1). Sam68 contains asymmetrical dimethylarginines near its proline motif P3 as assessed by using a novel asymmetrical dimethylarginine-specific antibody and mass spectrometry. Deletion of the methylation sites and the use of methylase inhibitors resulted in Sam68 accumulation in the cytoplasm. Sam68 was also detected in the cytoplasm of PRMT1-deficient embryonic stem cells. Although the cellular function of Sam68 is unknown, it has been shown to export unspliced human immunodeficiency virus RNAs. Cells treated with methylase inhibitors prevented the ability of Sam68 to export unspliced human immunodeficiency virus RNAs. Other K homology domain RNA binding proteins, including SLM-1, SLM-2, QKI-5, GRP33, and heteronuclear ribonucleoprotein K were also methylated in vivo. These findings demonstrate that RNA binding proteins are in vivo substrates for PRMT1, and their methylation is essential for their proper localization and function.     

Promiscuous modification of the nuclear poly(A)-binding protein by multiple protein-arginine methyltransferases does not affect the aggregation behavior

The mammalian nuclear poly(A)-binding protein, PABPN1, carries 13 asymmetrically dimethylated arginine residues in its C-terminal domain. By fractionation of cell extracts, we found that protein-arginine methyltransferases (PRMTs)-1, -3, and -6 are responsible for the modification of PABPN1. Recombinant PRMT1, -3, and -6 also methylated PABPN1. Our data suggest that these enzymes act on their own, and additional polypeptides are not involved in recognizing PABPN1 as a substrate. PRMT1 is the predominant methyltransferase acting on PABPN1. Nevertheless, PABPN1 was almost fully methylated in a Prmt1(-/-) cell line; thus, PRMT3 and -6 suffice for methylation. In contrast to PABPN1, the heterogeneous nuclear ribonucleoprotein (hnRNP) K is selectively methylated only by PRMT1. Efficient methylation of synthetic peptides derived from PABPN1 or hnRNP K suggested that PRMT1, -3, and -6 recognize their substrates by interacting with local amino acid sequences and not with additional domains of the substrates. However, the use of fusion proteins suggested that the inability of PRMT3 and -6 to modify hnRNP K is because of structural masking of the methyl-accepting amino acid sequences by neighboring domains. Mutations leading to intracellular aggregation of PABPN1 cause the disease oculopharyngeal muscular dystrophy. The C-terminal domain containing the methylated arginine residues is known to promote PAPBN1 self-association, and arginine methylation has been reported to inhibit self-association of an orthologous protein. Thus, arginine methylation might be relevant for oculopharyngeal muscular dystrophy. However, in two different types of assays we have been unable to detect any effect of arginine methylation on the aggregation of bovine PABPN1.     

A role for the arginine methylation of Rad9 in checkpoint control and cellular sensitivity to DNA damage

The genome stability is maintained by coordinated action of DNA repairs and checkpoints, which delay progression through the cell cycle in response to DNA damage. Rad9 is conserved from yeast to human and functions in cell cycle checkpoint controls. Here, a regulatory mechanism for Rad9 function is reported. In this study Rad9 has been found to interact with and be methylated by protein arginine methyltransferase 5 (PRMT5). Arginine methylation of Rad9 plays a critical role in S/M and G2/M cell cycle checkpoints. The activation of the Rad9 downstream checkpoint effector Chk1 is impaired in cells only expressing a mutant Rad9 that cannot be methylated. Additionally, Rad9 methylation is also required for cellular resistance to DNA damaging stresses. In summary, we uncovered that arginine methylation is important for regulation of Rad9 function, and thus is a major element for maintaining genome integrity.     

Arginine methylation regulates DNA polymerase beta

Alterations in DNA repair lead to genomic instability and higher risk of cancer. DNA base excision repair (BER) corrects damaged bases, apurinic sites, and single-strand DNA breaks. Here, a regulatory mechanism for DNA polymerase beta (Pol beta) is described. Pol beta was found to form a complex with the protein arginine methyltransferase 6 (PRMT6) and was specifically methylated in vitro and in vivo. Methylation of Pol beta by PRMT6 strongly stimulated DNA polymerase activity by enhancing DNA binding and processivity, while single nucleotide insertion and dRP-lyase activity were not affected. Two residues, R83 and R152, were identified in Pol beta as the sites of methylation by PRMT6. Genetic complementation of Pol beta knockout cells with R83/152K mutant revealed the importance of these residues for the cellular resistance to DNA alkylating agent. Based on our findings, we propose that PRMT6 plays a role as a regulator of BER.     

The Testis-Specific Factor CTCFL Cooperates with the Protein Methyltransferase PRMT7 in H19 Imprinting Control Region Methylation

Expression of imprinted genes is restricted to a single parental allele as a result of epigenetic regulation—DNA methylation and histone modifications. Igf2/H19 is a reciprocally imprinted locus exhibiting paternal Igf2 and maternal H19 expression. Their expression is regulated by a paternally methylated imprinting control region (ICR) located between the two genes. Although the de novo DNA methyltransferases have been shown to be necessary for the establishment of ICR methylation, the mechanism by which they are targeted to the region remains unknown. We demonstrate that CTCFL/BORIS, a paralog of CTCF, is an ICR-binding protein expressed during embryonic male germ cell development, coinciding with the timing of ICR methylation. PRMT7, a protein arginine methyltransferase with which CTCFL interacts, is also expressed during embryonic testis development. Symmetrical dimethyl arginine 3 of histone H4, a modification catalyzed by PRMT7, accumulates in germ cells during this developmental period. This modified histone is also found enriched in both H19 ICR and Gtl2 differentially methylated region (DMR) chromatin of testis by chromatin immunoprecipitation (ChIP) analysis. In vitro studies demonstrate that CTCFL stimulates the histone-methyltransferase activity of PRMT7 via interactions with both histones and PRMT7. Finally, H19 ICR methylation is demonstrated by nuclear co-injection of expression vectors encoding CTCFL, PRMT7, and the de novo DNA methyltransferases, Dnmt3a, -b and -L, in Xenopus oocytes. These results suggest that CTCFL and PRMT7 may play a role in male germline imprinted gene methylation.     

The testis-specific factor CTCFL cooperates with the protein methyltransferase PRMT7 in H19 imprinting control region methylation

Expression of imprinted genes is restricted to a single parental allele as a result of epigenetic regulation—DNA methylation and histone modifications. Igf2/H19 is a reciprocally imprinted locus exhibiting paternal Igf2 and maternal H19 expression. Their expression is regulated by a paternally methylated imprinting control region (ICR) located between the two genes. Although the de novo DNA methyltransferases have been shown to be necessary for the establishment of ICR methylation, the mechanism by which they are targeted to the region remains unknown. We demonstrate that CTCFL/BORIS, a paralog of CTCF, is an ICR-binding protein expressed during embryonic male germ cell development, coinciding with the timing of ICR methylation. PRMT7, a protein arginine methyltransferase with which CTCFL interacts, is also expressed during embryonic testis development. Symmetrical dimethyl arginine 3 of histone H4, a modification catalyzed by PRMT7, accumulates in germ cells during this developmental period. This modified histone is also found enriched in both H19 ICR and Gtl2 differentially methylated region (DMR) chromatin of testis by chromatin immunoprecipitation (ChIP) analysis. In vitro studies demonstrate that CTCFL stimulates the histone-methyltransferase activity of PRMT7 via interactions with both histones and PRMT7. Finally, H19 ICR methylation is demonstrated by nuclear co-injection of expression vectors encoding CTCFL, PRMT7, and the de novo DNA methyltransferases, Dnmt3a, -b and -L, in Xenopus oocytes. These results suggest that CTCFL and PRMT7 may play a role in male germline imprinted gene methylation.     

The Nijmegen breakage syndrome gene and its role in genome stability

NBS1 is the key regulator of the RAD50/MRE11/NBS1 (R/M/N) protein complex, a sensor and mediator for cellular DNA damage response. NBS1 potentiates the enzymatic activity of MRE11 and directs the R/M/N complex to sites of DNA damage, where it forms nuclear foci by interacting with phosphorylated H2AX. The R/M/N complex also activates the ATM kinase, which is a major kinase involved in the activation of DNA damage signal pathways. The ATM requires the R/M/N complex for its own activation following DNA damage, and for conformational change to develop a high affinity for target proteins. In addition, association of NBS1 with PML, the promyelocytic leukemia protein, is required to form nuclear bodies, which have various functions depending on their location and composition. These nuclear bodies function not only in response to DNA damage, but are also involved in telomere maintenance when they are located on telomeres. In this review, we describe the role of NBS1 in the maintenance of genetic stability through the activation of cell-cycle checkpoints, DNA repair, and protein relocation.     

Arginine methylation of ribosomal protein S3 affects ribosome assembly

The human ribosomal protein S3 (rpS3), a component of the 40S small subunit in the ribosome, is a known multi-functional protein with roles in DNA repair and apoptosis. We recently found that the arginine residue(s) of rpS3 are methylated by protein arginine methyltransferase 1 (PRMT1). In this paper, we confirmed the arginine methylation of rpS3 protein both in vitro and in vivo. The sites of arginine methylation are located at amino acids 64, 65 and 67. However, mutant rpS3 (3RA), which cannot be methylated at these sites, cannot be transported into the nucleolus and subsequently incorporated into the ribosome. Our results clearly show that arginine methylation of rpS3 plays a critical role in its import into the nucleolus, as well as in small subunit assembly of the ribosome.