FACS-based genome-wide CRISPR screens define key regulators of DNA damage signaling pathways.

DNA damage-activated signaling pathways are critical for coordinating multiple cellular processes, which must be tightly regulated to maintain genome stability. To provide a comprehensive and unbiased perspective of DNA damage response (DDR) signaling pathways, we performed 30 fluorescence-activated cell sorting (FACS)-based genome-wide CRISPR screens in human cell lines with antibodies recognizing distinct endogenous DNA damage signaling proteins to identify critical regulators involved in DDR. We discovered that proteasome-mediated processing is an early and prerequisite event for cells to trigger camptothecin- and etoposide-induced DDR signaling. Furthermore, we identified PRMT1 and PRMT5 as modulators that regulate ATM protein level. Moreover, we discovered that GNB1L is a key regulator of DDR signaling via its role as a co-chaperone specifically regulating PIKK proteins. Collectively, these screens offer a rich resource for further investigation of DDR, which may provide insight into strategies of targeting these DDR pathways to improve therapeutic outcomes.

PRMT6 methylation of RCC1 regulates mitosis, tumorigenicity, and radiation response of glioblastoma stem cells.

Aberrant cell proliferation is a hallmark of cancer, including glioblastoma (GBM). Here we report that protein arginine methyltransferase (PRMT) 6 activity is required for the proliferation, stem-like properties, and tumorigenicity of glioblastoma stem cells (GSCs), a subpopulation in GBM critical for malignancy. We identified a casein kinase 2 (CK2)-PRMT6-regulator of chromatin condensation 1 (RCC1) signaling axis whose activity is an important contributor to the stem-like properties and tumor biology of GSCs. CK2 phosphorylates and stabilizes PRMT6 through deubiquitylation, which promotes PRMT6 methylation of RCC1, which in turn is required for RCC1 association with chromatin and activation of RAN. Disruption of this pathway results in defects in mitosis. EPZ020411, a specific small-molecule inhibitor for PRMT6, suppresses RCC1 arginine methylation and improves the cytotoxic activity of radiotherapy against GSC brain tumor xenografts. This study identifies a CK2α-PRMT6-RCC1 signaling axis that can be therapeutically targeted in the treatment of GBM.

Arginine monomethylation by PRMT7 controls MAVS-mediated antiviral innate immunity.

Accurate control of innate immune responses is required to eliminate invading pathogens and simultaneously avoid autoinflammation and autoimmune diseases. Here, we demonstrate that arginine monomethylation precisely regulates the mitochondrial antiviral-signaling protein (MAVS)-mediated antiviral response. Protein arginine methyltransferase 7 (PRMT7) forms aggregates to catalyze MAVS monomethylation at arginine residue 52 (R52), attenuating its binding to TRIM31 and RIG-I, which leads to the suppression of MAVS aggregation and subsequent activation. Upon virus infection, aggregated PRMT7 is disabled in a timely manner due to automethylation at arginine residue 32 (R32), and SMURF1 is recruited to PRMT7 by MAVS to induce proteasomal degradation of PRMT7, resulting in the relief of PRMT7 suppression of MAVS activation. Therefore, we not only reveal that arginine monomethylation by PRMT7 negatively regulates MAVS-mediated antiviral signaling in vitro and in vivo but also uncover a mechanism by which PRMT7 is tightly controlled to ensure the timely activation of antiviral defense.

Molecular basis for substrate recruitment to the PRMT5 methylosome.

PRMT5 is an essential arginine methyltransferase and a therapeutic target in MTAP-null cancers. PRMT5 uses adaptor proteins for substrate recruitment through a previously undefined mechanism. Here, we identify an evolutionarily conserved peptide sequence shared among the three known substrate adaptors (CLNS1A, RIOK1, and COPR5) and show that it is necessary and sufficient for interaction with PRMT5. We demonstrate that PRMT5 uses modular adaptor proteins containing a common binding motif for substrate recruitment, comparable with other enzyme classes such as kinases and E3 ligases. We structurally resolve the interface with PRMT5 and show via genetic perturbation that it is required for methylation of adaptor-recruited substrates including the spliceosome, histones, and ribosomal complexes. Furthermore, disruption of this site affects Sm spliceosome activity, leading to intron retention. Genetic disruption of the PRMT5-substrate adaptor interface impairs growth of MTAP-null tumor cells and is thus a site for development of therapeutic inhibitors of PRMT5.

MST2 methylation by PRMT5 inhibits Hippo signaling and promotes pancreatic cancer progression.

The Hippo signaling axis is a tumor suppressor pathway that is activated by various extra-pathway factors to regulate cell differentiation and organ development. Recent studies have reported that autophosphorylation of the core kinase cassette stimulates activation of the Hippo signaling cascade. Here, we demonstrate that protein arginine methyltransferase 5 (PRMT5) contributes to inactivation of the Hippo signaling pathway in pancreatic cancer. We show that the Hippo pathway initiator serine/threonine kinase 3 (STK3, also known as MST2) of Hippo signaling pathway can be symmetrically di-methylated by PRMT5 at arginine-461 (R461) and arginine-467 (R467) in its SARAH domain. Methylation suppresses MST2 autophosphorylation and kinase activity by blocking its homodimerization, thereby inactivating Hippo signaling pathway in pancreatic cancer. Moreover, we also show that the specific PRMT5 inhibitor GSK3326595 re-activates the dysregulated Hippo signaling pathway and inhibits the growth of human pancreatic cancer xenografts in immunodeficient mice, thus suggesting potential clinical application of PRMT5 inhibitors in pancreatic cancer.

Asymmetric arginine dimethylation of cytosolic RNA and DNA sensors by PRMT3 attenuates antiviral innate immunity.

The cytosolic RNA and DNA sensors initiate type I interferon signaling when binding to RNA or DNA. To effectively protect the host against virus infection and concomitantly avoid excessive interferonopathy at resting states, these sensors must be tightly regulated. However, the key molecular mechanisms regulating these sensors' activation remain elusive. Here, we identify PRMT3, a type I protein arginine methyltransferase, as a negative regulator of cytosolic RNA and DNA sensors. PRMT3 interacts with RIG-I, MDA5, and cGAS and catalyzes asymmetric dimethylation of R730 on RIG-I, R822 on MDA5, and R111 on cGAS. These modifications reduce RNA-binding ability of RIG-I and MDA5 as well as DNA-binding ability and oligomerization of cGAS, leading to the inhibition of downstream type I interferon production. Furthermore, mice with loss of one copy of Prmt3 or in vivo treatment of the PRMT3 inhibitor, SGC707, are more resistant to RNA and DNA virus infection. Our findings reveal an essential role of PRMT3 in the regulation of antiviral innate immunity and give insights into the molecular regulation of cytosolic RNA and DNA sensors' activation.

MBD2a-NuRD binds to the methylated γ-globin gene promoter and uniquely forms a complex required for silencing of HbF expression.

During human development, there is a switch in the erythroid compartment at birth that results in silencing of expression of fetal hemoglobin (HbF). Reversal of this silencing has been shown to be effective in overcoming the pathophysiologic defect in sickle cell anemia. Among the many transcription factors and epigenetic effectors that are known to mediate HbF silencing, two of the most potent are BCL11A and MBD2-NuRD. In this report, we present direct evidence that MBD2-NuRD occupies the γ-globin gene promoter in adult erythroid cells and positions a nucleosome there that results in a closed chromatin conformation that prevents binding of the transcriptional activator, NF-Y. We show that the specific isoform, MBD2a, is required for the formation and stable occupancy of this repressor complex that includes BCL11A, MBD2a-NuRD, and the arginine methyltransferase, PRMT5. The methyl cytosine binding preference and the arginine-rich (GR) domain of MBD2a are required for high affinity binding to methylated γ-globin gene proximal promoter DNA sequences. Mutation of the methyl cytosine-binding domain (MBD) of MBD2 results in a variable but consistent loss of γ-globin gene silencing, in support of the importance of promoter methylation. The GR domain of MBD2a is also required for recruitment of PRMT5, which in turn results in placement of the repressive chromatin mark H3K8me2s at the promoter. These findings support a unified model that integrates the respective roles of BCL11A, MBD2a-NuRD, PRMT5, and DNA methylation in HbF silencing.

Combined inhibition of MTAP and MAT2a mimics synthetic lethality in tumor models via PRMT5 inhibition.

Homozygous 5'-methylthioadenosine phosphorylase (MTAP) deletions occur in approximately 15% of human cancers. Co-deletion of MTAP and methionine adenosyltransferase 2 alpha (MAT2a) induces a synthetic lethal phenotype involving protein arginine methyltransferase 5 (PRMT5) inhibition. MAT2a inhibitors are now in clinical trials for genotypic MTAP-/- cancers, however the MTAP-/- genotype represents fewer than 2% of human colorectal cancers (CRCs), limiting the utility of MAT2a inhibitors in these and other MTAP+/+ cancers. Methylthio-DADMe-immucillin-A (MTDIA) is a picomolar transition state analog inhibitor of MTAP that renders cells enzymatically MTAP-deficient to induce the MTAP-/- phenotype. Here, we demonstrate that MTDIA and MAT2a inhibitor AG-270 combination therapy mimics synthetic lethality in MTAP+/+ CRC cell lines with similar effects in mouse xenografts and without adverse histology on normal tissues. Combination treatment is synergistic with a 104-fold increase in drug potency for inhibition of CRC cell growth in culture. Combined MTDIA and AG-270 decreases S-adenosyl-L-methionine and increases 5'-methylthioadenosine in cells. The increased intracellular methylthioadenosine:S-adenosyl-L-methionine ratio inhibits PRMT5 activity, leading to cellular arrest and apoptotic cell death by causing MDM4 alternative splicing and p53 activation. Combination MTDIA and AG-270 treatment differs from direct inhibition of PRMT5 by GSK3326595 by avoiding toxicity caused by cell death in the normal gut epithelium induced by the PRMT5 inhibitor. The combination of MTAP and MAT2a inhibitors expands this synthetic lethal approach to include MTAP+/+ cancers, especially the remaining 98% of CRCs without the MTAP-/- genotype.

m6 A deposition is regulated by PRMT1-mediated arginine methylation of METTL14 in its disordered C-terminal region.

The N6-methyladenosine (m6 A) RNA modification serves crucial functions in RNA metabolism; however, the molecular mechanisms underlying the regulation of m6 A are not well understood. Here, we establish arginine methylation of METTL14, a component of the m6 A methyltransferase complex, as a novel pathway that controls m6 A deposition in mammalian cells. Specifically, protein arginine methyltransferase 1 (PRMT1) interacts with, and methylates the intrinsically disordered C terminus of METTL14, which promotes its interaction with RNA substrates, enhances its RNA methylation activity, and is crucial for its interaction with RNA polymerase II (RNAPII). Mouse embryonic stem cells (mESCs) expressing arginine methylation-deficient METTL14 exhibit significantly reduced global m6 A levels. Transcriptome-wide m6 A analysis identified 1,701 METTL14 arginine methylation-dependent m6 A sites located in 1,290 genes involved in various cellular processes, including stem cell maintenance and DNA repair. These arginine methylation-dependent m6 A sites are associated with enhanced translation of genes essential for the repair of DNA interstrand crosslinks; thus, METTL14 arginine methylation-deficient mESCs are hypersensitive to DNA crosslinking agents. Collectively, these findings reveal important aspects of m6 A regulation and new functions of arginine methylation in RNA metabolism.

In vitro methylation of the U7 snRNP subunits Lsm11 and SmE by the PRMT5/MEP50/pICln methylosome.

U7 snRNP is a multisubunit endonuclease required for 3' end processing of metazoan replication-dependent histone pre-mRNAs. In contrast to the spliceosomal snRNPs, U7 snRNP lacks the Sm subunits D1 and D2 and instead contains two related proteins, Lsm10 and Lsm11. The remaining five subunits of the U7 heptameric Sm ring, SmE, F, G, B, and D3, are shared with the spliceosomal snRNPs. The pathway that assembles the unique ring of U7 snRNP is unknown. Here, we show that a heterodimer of Lsm10 and Lsm11 tightly interacts with the methylosome, a complex of the arginine methyltransferase PRMT5, MEP50, and pICln known to methylate arginines in the carboxy-terminal regions of the Sm proteins B, D1, and D3 during the spliceosomal Sm ring assembly. Both biochemical and cryo-EM structural studies demonstrate that the interaction is mediated by PRMT5, which binds and methylates two arginine residues in the amino-terminal region of Lsm11. Surprisingly, PRMT5 also methylates an amino-terminal arginine in SmE, a subunit that does not undergo this type of modification during the biogenesis of the spliceosomal snRNPs. An intriguing possibility is that the unique methylation pattern of Lsm11 and SmE plays a vital role in the assembly of the U7 snRNP.

PRMT4 interacts with NCOA4 to inhibit ferritinophagy in cisplatin-induced acute kidney injury.

Cisplatin-induced acute kidney injury (AKI) is commonly seen in the clinical practice, and ferroptosis, a type of non-apoptotic cell death, plays a pivotal role in it. Previous studies suggested that protein arginine methyltransferase 4 (PRMT4) was incorporated in various bioprocesses, but its role in renal injuries has not been investigated. Our present study showed that PRMT4 was highly expressed in renal proximal tubular cells, and it was downregulated in cisplatin-induced AKI. Besides, genetic disruption of PRMT4 exacerbated, while its overexpression attenuated, cisplatin-induced redox injuries in renal proximal epithelia. Mechanistically, our work showed that PRMT4 interacted with NCOA4 to inhibit ferritinophagy, a type of selective autophagy favoring lipid peroxidation to accelerate ferroptosis. Taken together, our study demonstrated that PRMT4 interacted with NCOA4 to attenuate ferroptosis in cisplatin-induced AKI, suggesting that PRMT4 might present as a new therapeutic target for cisplatin-related nephropathy.

HSV-1 reactivation results in post-herpetic neuralgia by upregulating Prmt6 and inhibiting cGAS-STING.

Chronic varicella zoster virus (VZV) infection induced neuroinflammatory condition is the critical pathology of post-herpetic neuralgia (PHN). The immune escape mechanism of VZV remains elusive. As to mice have no VZV infection receptor, herpes simplex virus type 1 (HSV-1) infection is a well established PHN mice model. Transcriptional expression analysis identified that the protein arginine methyltransferases 6 (Prmt6) was upregulated upon HSV-1 infection, which was further confirmed by immunofluorescence staining in spinal dorsal horn. Prmt6 deficiency decreased HSV-1-induced neuroinflammation and PHN by enhancing antiviral innate immunity and decreasing HSV-1 load in vivo and in vitro. Overexpression of Prmt6 in microglia dampened antiviral innate immunity and increased HSV-1 load. Mechanistically, Prmt6 methylated and inactivated STING, resulting in reduced phosphorylation of TANK binding kinase-1 (TBK1) and interferon regulatory factor 3 (IRF3), diminished production of type I interferon (IFN-I) and antiviral innate immunity. Furthermore, intrathecal or intraperitoneal administration of the Prmt6 inhibitor EPZ020411 decreased HSV-1-induced neuroinflammation and PHN by enhancing antiviral innate immunity and decreasing HSV-1 load. Our findings revealed that HSV-1 escapes antiviral innate immunity and results in PHN by upregulating Prmt6 expression and inhibiting the cGAS-STING pathway, providing novel insights and a potential therapeutic target for PHN.

Arginine Methylation: The Coming of Age.

Arginine methylation is a common post-translational modification functioning as an epigenetic regulator of transcription and playing key roles in pre-mRNA splicing, DNA damage signaling, mRNA translation, cell signaling, and cell fate decision. Recently, a wealth of studies using transgenic mouse models and selective PRMT inhibitors helped define physiological roles for protein arginine methyltransferases (PRMTs) linking them to diseases such as cancer and metabolic, neurodegenerative, and muscular disorders. This review describes the recent molecular advances that have been uncovered in normal and diseased mammalian cells.

Nitric Oxide Regulates Protein Methylation during Stress Responses in Plants.

Methylation and nitric oxide (NO)-based S-nitrosylation are highly conserved protein posttranslational modifications that regulate diverse biological processes. In higher eukaryotes, PRMT5 catalyzes Arg symmetric dimethylation, including key components of the spliceosome. The Arabidopsis prmt5 mutant shows severe developmental defects and impaired stress responses. However, little is known about the mechanisms regulating the PRMT5 activity. Here, we report that NO positively regulates the PRMT5 activity through S-nitrosylation at Cys-125 during stress responses. In prmt5-1 plants, a PRMT5C125S transgene, carrying a non-nitrosylatable mutation at Cys-125, fully rescues the developmental defects, but not the stress hypersensitive phenotype and the responsiveness to NO during stress responses. Moreover, the salt-induced Arg symmetric dimethylation is abolished in PRMT5C125S/prmt5-1 plants, correlated to aberrant splicing of pre-mRNA derived from a stress-related gene. These findings define a mechanism by which plants transduce stress-triggered NO signal to protein methylation machinery through S-nitrosylation of PRMT5 in response to environmental alterations.

PRMT5-Dependent Methylation of the TIP60 Coactivator RUVBL1 Is a Key Regulator of Homologous Recombination.

Protein post-translation modification plays an important role in regulating DNA repair; however, the role of arginine methylation in this process is poorly understood. Here we identify the arginine methyltransferase PRMT5 as a key regulator of homologous recombination (HR)-mediated double-strand break (DSB) repair, which is mediated through its ability to methylate RUVBL1, a cofactor of the TIP60 complex. We show that PRMT5 targets RUVBL1 for methylation at position R205, which facilitates TIP60-dependent mobilization of 53BP1 from DNA breaks, promoting HR. Mechanistically, we demonstrate that PRMT5-directed methylation of RUVBL1 is critically required for the acetyltransferase activity of TIP60, promoting histone H4K16 acetylation, which facilities 53BP1 displacement from DSBs. Interestingly, RUVBL1 methylation did not affect the ability of TIP60 to facilitate ATM activation. Taken together, our findings reveal the importance of PRMT5-mediated arginine methylation during DSB repair pathway choice through its ability to regulate acetylation-dependent control of 53BP1 localization.

Promising role of protein arginine methyltransferases in overcoming anti-cancer drug resistance.

Drug resistance remains a major challenge in cancer treatment, necessitating the development of novel strategies to overcome it. Protein arginine methyltransferases (PRMTs) are enzymes responsible for epigenetic arginine methylation, which regulates various biological and pathological processes, as a result, they are attractive therapeutic targets for overcoming anti-cancer drug resistance. The ongoing development of small molecules targeting PRMTs has resulted in the generation of chemical probes for modulating most PRMTs and facilitated clinical treatment for the most advanced oncology targets, including PRMT1 and PRMT5. In this review, we summarize various mechanisms underlying protein arginine methylation and the roles of specific PRMTs in driving cancer drug resistance. Furthermore, we highlight the potential clinical implications of PRMT inhibitors in decreasing cancer drug resistance. PRMTs promote the formation and maintenance of drug-tolerant cells via several mechanisms, including altered drug efflux transporters, autophagy, DNA damage repair, cancer stem cell-related function, epithelial-mesenchymal transition, and disordered tumor microenvironment. Multiple preclinical and ongoing clinical trials have demonstrated that PRMT inhibitors, particularly PRMT5 inhibitors, can sensitize cancer cells to various anti-cancer drugs, including chemotherapeutic, targeted therapeutic, and immunotherapeutic agents. Combining PRMT inhibitors with existing anti-cancer strategies will be a promising approach for overcoming anti-cancer drug resistance. Furthermore, enhanced knowledge of the complex functions of arginine methylation and PRMTs in drug resistance will guide the future development of PRMT inhibitors and may help identify new clinical indications.

ETD-Based Proteomic Profiling Improves Arginine Methylation Identification and Reveals Novel PRMT5 Substrates.

Protein arginine methylations are important post-translational modifications (PTMs) in eukaryotes, regulating many biological processes. However, traditional collision-based mass spectrometry methods inevitably cause neutral losses of methylarginines, preventing the deep mining of biologically important sites. Herein we developed an optimized mass spectrometry workflow based on electron-transfer dissociation (ETD) with supplemental activation for proteomic profiling of arginine methylation in human cells. Using symmetric dimethylarginine (sDMA) as an example, we show that the ETD-based optimized workflow significantly improved the identification and site localization of sDMA. Quantitative proteomics identified 138 novel sDMA sites as potential PRMT5 substrates in HeLa cells. Further biochemical studies on SERBP1, a newly identified PRMT5 substrate, confirmed the coexistence of sDMA and asymmetric dimethylarginine in the central RGG/RG motif, and loss of either methylation caused increased the recruitment of SERBP1 to stress granules under oxidative stress. Overall, our optimized workflow not only enabled the identification and localization of extensive, nonoverlapping sDMA sites in human cells but also revealed novel PRMT5 substrates whose sDMA may play potentially important biological functions.

PRMT7-Dependent Transcriptional Activation of Hmgb2 Aggravates Severe Acute Pancreatitis by Promoting Acsl1-Induced Ferroptosis.

Severe acute pancreatitis (SAP) is a highly fatal abdominal emergency, and its association with protein arginine methyltransferase 7 (PRMT7), the sole known type III enzyme responsible for the monomethylation of arginine residue, remains unexplored. In this study, we observe an increase in the PRMT7 levels in the pancreas of SAP mice and Cerulein-LPS-stimulated AR42J cells. Overexpression of Prmt7 exacerbated pancreatic damage in SAP, while the inhibition of PRMT7 improved SAP-induced pancreatic damage. Furthermore, PRMT7 overexpression promoted inflammation, oxidative stress, and ferroptosis during SAP. Mechanically, PRMT7 catalyzed monomethylation at histone H4 arginine 3 (H4R3me1) at the promoter region of high mobility group proteins 2 (HMGB2), thereby enhancing its transcriptional activity. Subsequently, HMGB2 facilitated Acyl CoA synthase long-chain family member 1 (ACSL1) transcription by binding to its promoter region, resulting in the activation of ferroptosis. Inhibition of PRMT7 effectively alleviated ferroptosis in Cerulein-LPS-induced AR42J cells by suppressing the HMGB2-ACSL1 pathway. Overall, our study reveals that PRMT7 plays a crucial role in promoting SAP through its regulation of the HMGB2-ACSL1 pathway to accelerate ferroptosis.

Activation of protein arginine methyltransferase 1 and subsequent extension of moth lifespan is effected by the ROS/JNK/CREB signaling axis.

Previous studies have demonstrated that high physiological levels of reactive oxygen species induce pupal diapause and extend lifespan in the moth Helicoverpa armigera. This has been shown to occur via protein arginine methyltransferase 1 (PRMT1) blockade of Akt-mediated phosphorylation of the transcription factor FoxO, after which activated FoxO promotes the initiation of diapause. However, it is unclear how PRMT1 is activated upstream of FoxO activity. Here, we show that high reactive oxygen species levels in the brains of H. armigera diapause-destined pupae activate the expression of c-Jun N-terminal kinase, which subsequently activates the transcription factor cAMP-response element binding protein. We show that cAMP-response element binding protein then directly binds to the PRMT1 promoter and upregulates its expression to prevent Akt-mediated FoxO phosphorylation and downstream FoxO nuclear localization. This novel finding that c-Jun N-terminal kinase promotes FoxO nuclear localization in a PRMT1-dependent manner to regulate pupal diapause reveals a complex regulatory mechanism in extending the healthspan of H. armigera.

Prmt5 promotes ciliated cell specification of airway epithelial progenitors via transcriptional inhibition of Tp63.

The epithelium of the pulmonary airway is composed of several distinct cell types that differentiate from common progenitor cells to provide defense against environmental insults. Epigenetic mechanisms regulating lineage differentiation of airway epithelial progenitors remain poorly understood. Protein arginine methyltransferase 5 (Prmt5) is a predominant type II arginine methyltransferase that methylates >85% of symmetric arginine residues. Here, we provide evidence for the function of Prmt5 in promoting ciliated cell fate specification of airway epithelial progenitors. We show that lung epithelial-specific deletion of Prmt5 resulted in a complete loss of ciliated cells, an increased number of basal cells, and ecotopic-expressed Tp63-Krt5+ putative cells in the proximal airway. We further identified that transcription factor Tp63 is a direct target of Prmt5, and Prmt5 inhibited Tp63 transcription expression through H4R3 symmetric dimethylation (H4R3sme2). Moreover, inhibition of Tp63 expression in Prmt5-deficient tracheal progenitors could partially restore the ciliated cell deficient phenotype. Together, our data support a model where Prmt5-mediated H4R3sme2 represses Tp63 expression to promote ciliated cell fate specification of airway progenitors.