RESUMO
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.
Assuntos
Arginina , Interferon Tipo I , Animais , Camundongos , RNA/genética , Antivirais/farmacologia , Imunidade Inata , DNA/genética , Nucleotidiltransferases/genética , Proteína-Arginina N-Metiltransferases/genéticaRESUMO
Arginine methylation catalyzed by protein arginine methyltransferases (PRMT) is a common post-translational modification in histone and nonhistone proteins, which regulates many cellular functions. Protein arginine methyltransferase 3 (prmt3), a type I arginine methyltransferase, has been shown to carry out the formation of stable monomethylarginine as an intermediate before the establishment of asymmetric dimethylarginine. To date, however, the role of PRMT3 in antiviral innate immunity has not been elucidated. This study showed that zebrafish prmt3 was upregulated by virus infection and that the overexpression of prmt3 suppressed cellular antiviral response. The PRMT3 inhibitor, SGC707, enhanced antiviral capability. Consistently, prmt3-null zebrafish were more resistant to Spring Viremia of Carp Virus (SVCV) and Grass Carp Reovirus (GCRV) infection. Further assays showed that the overexpression of prmt3 diminished the phosphorylation of irf3 and prmt3 interacted with rig-i. In addition, both zinc-finger domain and catalytic domain of prmt3 were required for the suppressive function of prmt3 on IFN activation. Our findings suggested that zebrafish prmt3 negatively regulated the antiviral responses, implicating the vital role of prmt3-or even arginine methylation-in antiviral innate immunity.
Assuntos
Antivirais/imunologia , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/imunologia , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Animais , Células Cultivadas , Histonas/genética , Histonas/imunologia , Imunidade Inata/genética , Imunidade Inata/imunologia , Isoquinolinas/imunologia , Metilação , Fosforilação/genética , Fosforilação/imunologia , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/imunologia , Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/genética , Infecções por Rhabdoviridae/imunologia , Regulação para Cima/genética , Regulação para Cima/imunologia , Viroses/genética , Viroses/imunologia , Viroses/virologia , Peixe-Zebra/virologia , Dedos de Zinco/genética , Dedos de Zinco/imunologiaRESUMO
Ribosomal (r)-proteins are generally viewed as ubiquitous, constitutive proteins that simply function to maintain ribosome integrity. However, findings in the past decade have led to the idea that r-proteins have evolved specialized functions beyond the ribosome. For example, the 40S ribosomal protein uS5 (RPS2) is known to form an extraribosomal complex with the protein arginine methyltransferase PRMT3 that is conserved from fission yeast to humans. However, the full scope of uS5's extraribosomal functions, including whether uS5 interacts with any other proteins, is not known. In this study, we identify the conserved zinc finger protein 277 (ZNF277) as a new uS5-associated protein by using quantitative proteomics approaches in human cells. As previously shown for PRMT3, we found that ZNF277 uses a C2H2-type zinc finger domain to recognize uS5. Analysis of protein-protein interactions in living cells indicated that the ZNF277-uS5 complex is found in the cytoplasm and the nucleolus. Furthermore, we show that ZNF277 and PRMT3 compete for uS5 binding, because overexpression of PRMT3 inhibited the formation of the ZNF277-uS5 complex, whereas depletion of cellular ZNF277 resulted in increased levels of uS5-PRMT3. Notably, our results reveal that ZNF277 recognizes nascent uS5 in the course of mRNA translation, suggesting cotranslational assembly of the ZNF277-uS5 complex. Our findings thus unveil an intricate network of evolutionarily conserved protein-protein interactions involving extraribosomal uS5, suggesting a key role for uS5 beyond the ribosome.
Assuntos
Nucléolo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Ribossômicas/metabolismo , Nucléolo Celular/genética , Citoplasma/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , Células HeLa , Humanos , Complexos Multiproteicos/genética , Proteína-Arginina N-Metiltransferases/genética , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/genéticaRESUMO
Protein arginine methyltransferases (PRMTs) in mammals play a role in various signaling pathways, such as virus infection, inflammasome responses, and cancer growth. While some PRMTs have been found to regulate interferon production in mammals, the mechanism in chickens remains to be fully understood. This study focused on investigating the function of chicken PRMTs. Our findings indicate that chicken PRMTs act as inhibitors of interferon production in response to dsRNA or MDA5 stimulation. Each PRMT is involved in different stages of interferon induction through the MDA5-MAVS-TBK1 pathway. Furthermore, we observed the colocalization of multiple PRMTs with the viral protein VP3 of infectious bursal disease virus (IBDV). Among the chicken PRMTs studied, PRMT3 was found to be widely expressed in various organs and its expression was upregulated during IBDV infection. Notably, PRMT3 supported IBDV replication, as demonstrated by ectopic expression and inhibition studies using SGC-707. Silencing of PRMT3 led to enhanced interferon production and inhibition of IBDV replication. This study provides novel insights into the role of chicken PRMTs, particularly PRMT3, in promoting IBDV replication by suppressing interferon signaling.
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Protein arginine methyltransferase 3 (PRMT3) plays an important role in gene regulation and a variety of cellular functions, thus, being a long sought-after therapeutic target for human cancers. Although a few PRMT3 inhibitors are developed to prevent the catalytic activity of PRMT3, there is little success in removing the cellular levels of PRMT3-deposited ω-NG,NG-asymmetric dimethylarginine (ADMA) with small molecules. Moreover, the non-enzymatic functions of PRMT3 remain required to be clarified. Here, the development of a first-in-class MDM2-based PRMT3-targeted Proteolysis Targeting Chimeras (PROTACs) 11 that selectively reduced both PRMT3 protein and ADMA is reported. Importantly, 11 inhibited acute leukemia cell growth and is more effective than PRMT3 inhibitor SGC707. Mechanism study shows that 11 induced global gene expression changes, including the activation of intrinsic apoptosis and endoplasmic reticulum stress signaling pathways, and the downregulation of E2F, MYC, oxidative phosphorylation pathways. Significantly, the combination of 11 and glycolysis inhibitor 2-DG has a notable synergistic antiproliferative effect by further reducing ATP production and inducing intrinsic apoptosis, thus further highlighting the potential therapeutic value of targeted PRMT3 degradation. These data clearly demonstrated that degrader 11 is a powerful chemical tool for investigating PRMT3 protein functions.
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Post-translational modification (PTM) of proteins increases proteome diversity, which is critical for maintaining cellular homeostasis. The importance of protein methylation in the regulation of diverse biological processes has been highlighted in the past decades. Methylation of the arginine residue on proteins is catalyzed by members of the protein arginine methyltransferase (PRMT) family. PRMTs play indispensable roles in various pathways that regulate cancer development, progression, and drug response. In this review, we discuss the role of PRMT3, a member of the PRMT family, in controlling oncogenic processes. Additionally, the effects of PRMT3 on the methylation of regulatory proteins involved in transcription, post-transcriptional control, ribosomal maturation, translation, biological synthesis, and metabolic signaling are summarized. Moreover, recent progresses in the development of PRMT3 inhibitors are introduced. Overall, this review highlights the importance of PRMT3 in tumorigenesis and discusses the underlying mechanisms by which PRMT3 modulates cellular metabolism and gene expression. These results also provide a molecular basis for therapeutic modalities by targeting PRMT3.
Assuntos
Neoplasias , Proteína-Arginina N-Metiltransferases , Humanos , Arginina , Expressão Gênica , Metilação , Neoplasias/genética , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
Protein arginine methyltransferase (PRMT) plays essential roles in tumor initiation and progression, but its underlying mechanisms in the treatment sensitivity of endometrial cancer (EC) remain unclear and warrant further investigation. Here, a comprehensive analysis of the Cancer Genome Atlas database and Clinical Proteomic Tumor Analysis Consortium database identifies that PRMT3 plays an important role in EC. Specifically, further experiments show that PRMT3 inhibition enhances the susceptibility of EC cells to ferroptosis. Mechanistically, PRMT3 interacts with Methyltransferase 14 (METTL14) and is involved in its arginine methylation. In addition, PRMT3 inhibition-mediated METTL14 overexpression promotes methylation modification via an m6 A-YTHDF2-dependent mechanism, reducing Glutathione peroxidase 4 (GPX4) mRNA stability, increasing lipid peroxidation levels, and accelerating ferroptosis. Notably, combined PRMT3 blockade and anti-PD-1 therapy display more potent antitumor effects by accelerating ferroptosis in cell-derived xenograft models. The specific PRMT3 inhibitor SGC707 exerts the same immunotherapeutic sensitizing effect in a patient-derived xenograft model. Notably, blocking PRMT3 improves tumor suppression in response to cisplatin and radiation therapy. Altogether, this work demonstrates that PRMT3 depletion is a promising target for EC.
Assuntos
Arginina , Neoplasias do Endométrio , Humanos , Feminino , Arginina/metabolismo , Proteômica , Metilação , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/genética , Metiltransferases/metabolismoRESUMO
Ribosomal proteins are fundamental components of the ribosomes in all living cells. The ribosomal protein uS5 (Rps2) is a stable component of the small ribosomal subunit within all three domains of life. In addition to its interactions with proximal ribosomal proteins and rRNA inside the ribosome, uS5 has a surprisingly complex network of evolutionarily conserved non-ribosome-associated proteins. In this review, we focus on a set of four conserved uS5-associated proteins: the protein arginine methyltransferase 3 (PRMT3), the programmed cell death 2 (PDCD2) and its PDCD2-like (PDCD2L) paralog, and the zinc finger protein, ZNF277. We discuss recent work that presents PDCD2 and homologs as a dedicated uS5 chaperone and PDCD2L as a potential adaptor protein for the nuclear export of pre-40S subunits. Although the functional significance of the PRMT3-uS5 and ZNF277-uS5 interactions remain elusive, we reflect on the potential roles of uS5 arginine methylation by PRMT3 and on data indicating that ZNF277 and PRMT3 compete for uS5 binding. Together, these discussions highlight the complex and conserved regulatory network responsible for monitoring the availability and the folding of uS5 for the formation of 40S ribosomal subunits and/or the role of uS5 in potential extra-ribosomal functions.
Assuntos
Amigos , Proteínas Ribossômicas , Humanos , Proteínas Ribossômicas/metabolismo , Proteína-Arginina N-Metiltransferases/química , Ribossomos/metabolismo , RNA Ribossômico/metabolismo , Proteínas Reguladoras de Apoptose/metabolismoRESUMO
BACKGROUND: Protein arginine methylation has emerged a pivotal role in cancer progression. However, the role of protein arginine methyltransferase 3 (PRMT3) in hepatocellular carcinoma (HCC) remains unknown. METHODS: The expression pattern of PRMT3 in HCC was analysed using quantitative real-time-polymerase chain reaction (qRT-PCR), Western blotting and immunohistochemistry assays. Loss- and gain-of-function experiments were carried out to determine the oncogenic role of PRMT3 in HCC. Glucose consumption and lactate production assays, seahorse bioscience, mass spectrometry, co-immunoprecipitation, metabonomic analysis and site-specific mutation experiments were used to explore the underlying molecular mechanisms. Furthermore, a xenograft mouse model was established to investigate the effects of PRMT3 and its inhibitor, SGC707, treatment on tumour growth in vivo. RESULTS: The expression of PRMT3 was significantly upregulated in HCC, with high expression of which correlated with poor prognosis. PRMT3 knockdown led to the decrease in proliferation, glycolysis of HCC cells and tumour growth, whilst its overexpression showed opposite results. The catalytic activity of PRMT3 was important in mediating these biological processes. Mechanistically, our data showed that PRMT3 interacted with and mediated asymmetric dimethylarginine (ADMA) modification of lactate dehydrogenase A (LDHA) at arginine 112 (R112). Compared with LDHA-wild-type (LDHA-WT) cells, LDHA-R112K-mutant-expressing HCC cells exhibited a decrease in lactate dehydrogenase (LDH) activity, HCC cell glycolysis and proliferation. Furthermore, the administration of SGC707, a selective inhibitor of PRMT3, disrupted the PRMT3-mediated LDHA methylation and abolished PRMT3-induced HCC glycolysis and tumour growth. CONCLUSIONS: Our results suggested a novel oncogenic role of PRMT3 in HCC, and it could be a promising therapeutic target for HCC by linking post-translational modification and cancer metabolism.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Glicólise/efeitos dos fármacos , L-Lactato Desidrogenase/farmacologia , Proteína-Arginina N-Metiltransferases/farmacologia , Animais , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Proliferação de Células/efeitos dos fármacos , China , Modelos Animais de Doenças , Histologia/instrumentação , Histologia/tendências , Humanos , L-Lactato Desidrogenase/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Metilação/efeitos dos fármacos , Camundongos , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
The incidence rates of colorectal cancer have been increasing in the last decades, yet the overall survival rate is still not ideal. There is a need to further investigate detailed mechanism for colorectal cancer tumorigenesis. The biological function of protein arginine methyltransferases 3 (PRMT3) is seldom studied in tumorigenesis. Here, we attempted to elucidate the link between PRMT3 and tumorigenesis in colorectal cancer. Results revealed that PRMT3 was upregulated in colorectal cancer. Besides, PRMT3 overexpression promoted colorectal cancer cell proliferation, migration, and invasion. Regarding mechanism for colorectal cancer tumorigenesis, PRMT3 stabilized C-MYC and the pro-tumorigenesis function of PRMT3 was dependent on C-MYC. Clinically, these findings might provide a novel therapeutical treatment strategy for colorectal cancer.
Assuntos
Neoplasias Colorretais/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Carcinogênese/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Metilação , Invasividade Neoplásica/genética , Proteína-Arginina N-Metiltransferases/fisiologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Ribossômicas/metabolismoRESUMO
Through experiments to testify a candidate novel miRNA previously discovered by us is a real miRNA and involved in cartilage development. DESIGN: The miR-novel and the newly hairpin miRNA transcribed sequence (pre-miR-novel) was verified as a genuinely existing miRNA by northern blotting. The predicted secondary structure, sequence alignment and targets of pre-miR-novel were performed by "RNAstructure 5.3" program, LASTN2.8.0+/miRbase22 program and RNA hybird program, respective. GO/KEGG pathway analysis also were performed. The miR-novel expression in cartilage tissue during development was detected by RT-qPCR and dot blotting. The chondrocyte differentiation model was established to examine whether miR-novel is involved in cartilage development. The regulation of PRMT3 expression by novel miRNA was determined with the luciferase reporter gene assay and Western blotting after novel miRNA mimic or inhibitor transfection. RESULTS: It's potential role in specifically regulating rodent cartilage development and associated cellular processes. Furthermore, the expression of protein arginine N-methyltransferase 3 (PRMT3), as a predicted target of the novel miRNA, was found consistently downregulated at rat cartilage during developmental stages and RCJ3.1C5.18 (C5.18) cells during the proliferating and hypertrophic phases of the cartilage development, where the miR-novel expression was significantly up-regulated. Both the dual-luciferase reporter gene assay and the up- or down-regulation of miR-novel suggest that the later can specifically bind with the Prmt3 3'-UTR. CONCLUSION: Overall, this study provides the first comprehensive evidence that a genuine cartilage-specific novel miRNA directly targets PRMT3 and may regulate multitudinous cellular processes and signal transduction during cartilage development.
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Iron is an essential trace metal for all biological processes and plays a role in almost every aspect of body growth. Previously, we found that iron-depletion downregulated the expression of proteins, arginine methyltransferase-1 and 3 (PRMT1 and PRMT3), by an iron-specific chelator, deferoxamine (DFO), in rat liver FAO cell line using DNA microarray analysis (unpublished data). However, regulatory mechanisms underlying the association between iron deficiency and PRMT expression are unclear in vitro and in vivo. In the present study, we revealed that the treatment of cells with two iron-specific chelators, DFO and deferasirox (DFX), downregulated the gene and protein expression of PRMT1 and 3 as compared with the untreated cells. Subsequently, DFO and DFX treatments decreased protein methylation. Importantly, these effects were attenuated by a holo-transferrin treatment. Furthermore, weanling Wistar-strain rats were fed a control diet or an iron-deficient diet for 4 weeks. Dietary iron deficiency was found to decrease the concentration of hemoglobin and liver iron while increasing the heart weight. PRMT and protein methylation levels were also significantly reduced in the iron-deficient group as compared to the control group. To our knowledge, this is the first study to demonstrate that PRMT levels and protein methylation are reduced in iron-deficient models, in vitro and in vivo.
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Pancreatic cancer is poorly responsive to chemotherapy due to intrinsic or acquired resistance. Our previous study showed that epigenetic modifying enzymes including protein arginine methyltransferase 3 (PRMT3) are dysregulated in gemcitabine (GEM)-resistant pancreatic cancer cells. Here, we attempt to elucidate the role of PRMT3 in chemoresistance. Overexpression of PRMT3 led to increased resistance to GEM in pancreatic cancer cells, whereas reduction of PRMT3 restored GEM sensitivity in resistant cells. We identified a novel PRMT3 target, ATP-binding cassette subfamily G member 2 (ABCG2), which is known to play a critical role in drug resistance. PRMT3 overexpression upregulated ABCG2 expression by increasing its mRNA stability. Mass spectrometric analysis identified hnRNPA1 as a PRMT3 interacting protein, and methylation of hnRNPA1 at R31 by PRMT3 in vivo and in vitro. The expression of methylation-deficient hnRNPA1-R31K mutant reduced the RNA binding activity of hnRNPA1 and the expression of ABCG2 mRNA. Taken together, this provides the first evidence that PRMT3 methylates the RNA recognition motif (RRM) of hnRNPA1 and promotes the binding between hnRNPA1 and ABCG2 to enhance drug resistance. Inhibition of PRMT3 could be a novel strategy for the treatment of GEM-resistant pancreatic cancer.
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Irisin is a newly identified myokine related to exercise and the browning of white fat. Recently, it was reported that irisin serum levels are associated with intrahepatic triglyceride content, suggesting that it might have an important role in the liver. The aim of this study was to determine the role of irisin in hepatocytes. Specifically, the effect of recombinant irisin on palmitic acid (PA)-induced lipogenesis and its related signal pathways were examined in AML12 cells and mouse primary hepatocytes. In the present study, we observed the presence of irisin inside the cells in response to the treatment of recombinant irisin by flow cytometry and cell imaging technique. Recombinant irisin significantly inhibited the PA-induced increase in lipogenic markers ACC and FAS at the mRNA and protein levels, and prevented the PA-induced lipid accumulation in hepatocytes. Additionally, irisin inhibited the PA-induced increase in the expression, nuclear localization, and transcriptional activities of the master regulators of lipogenesis (LXRα and SREBP-1c). Moreover, irisin attenuated PA-induced oxidative stress, which was confirmed by measuring the expression of inflammatory markers (NFκB, COX-2, p38 MAPK, TNF, IL-6) and superoxide indicator (dihydroethidium). The preventive effects of irisin against lipogenesis and oxidative stress were mediated by the inhibition of protein arginine methyltransferase-3 (PRMT3). These findings suggested that irisin might have a beneficial role in the prevention of hepatic steatosis by altering the expression of lipogenic genes and attenuating oxidative stress in a PRMT3 dependent manner.
Assuntos
Fígado Gorduroso/metabolismo , Fibronectinas/metabolismo , Hepatócitos/metabolismo , Animais , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Fígado Gorduroso/prevenção & controle , Fibronectinas/genética , Hepatócitos/patologia , Receptores X do Fígado , Camundongos , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ácido Palmítico/toxicidade , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismoRESUMO
Objective To construct plasmids for knock-out of protein arginine methyltransferase 3 (prmt3) gene using CRISPR/Cas9 gene editing method and examine the effect of prmt3 knockout on the proliferation of human non-small cell lung cancer(NSCLC)A549 cells.Methods Synthesized sgRNA oligos targeting prmt3 gene were cloned into LentiCRISPR vector and positive constructs confirmed by sequencing later .After infection with the packaged virus , A549 cells were screened with puromycin , and then the single clones were isolated .The protein level of PRMT3 in individual cell clones was analyzed with Western blot . Biological assay of clone formation , wound healing , flow cytometry assay and mass spectrometry ( MS) analysis were used to compare cellular proliferation behavior changes between control cells and cells with prmt3 gene knockout .Results The LentiCRISPR plasmids targeting prmt3 gene were confirmed by sequencing , and the PRMT3 protein level was significantly decreased in PRMT 3 KO cells compared with control cells .Depletion of PRMT3 promoted cell proliferation and led to cell cycle arrest at G 2/M phase, but had no influence on cell migration .Besides, some PRMT3 substrate candidates were identified with mass spectrum assays .Conclusion A549 cells with prmt3 gene knockout based on CRISPR/Cas9 are successfully established .PRMT3 can regulate cell cycle and proliferation .