SART3 reads methylarginine-marked glycine- and arginine-rich motifs.

Glycine- and arginine-rich (GAR) motifs, commonly found in RNA-binding and -processing proteins, can be symmetrically (SDMA) or asymmetrically (ADMA) dimethylated at the arginine residue by protein arginine methyltransferases. Arginine-methylated protein motifs are usually read by Tudor domain-containing proteins. Here, using a GFP-Trap, we identify a non-Tudor domain protein, squamous cell carcinoma antigen recognized by T cells 3 (SART3), as a reader for SDMA-marked GAR motifs. Structural analysis and mutagenesis of SART3 show that aromatic residues lining a groove between two adjacent aromatic-rich half-a-tetratricopeptide (HAT) repeat domains are essential for SART3 to recognize and bind to SDMA-marked GAR motif peptides, as well as for the interaction between SART3 and the GAR-motif-containing proteins fibrillarin and coilin. Further, we show that the loss of this reader ability affects RNA splicing. Overall, our findings broaden the range of potential SDMA readers to include HAT domains.

Dysregulation of arginine methylation in tumorigenesis.

Protein methylation, similar to DNA methylation, primarily involves post-translational modification (PTM) targeting residues of nitrogen-containing side-chains and other residues. Protein arginine methylation, occurred on arginine residue, is mainly mediated by protein arginine methyltransferases (PRMTs), which are ubiquitously present in a multitude of organisms and are intricately involved in the regulation of numerous biological processes. Specifically, PRMTs are pivotal in the process of gene transcription regulation, and protein function modulation. Abnormal arginine methylation, particularly in histones, can induce dysregulation of gene expression, thereby leading to the development of cancer. The recent advancements in modification mediated by PRMTs and cancer research have had a profound impact on our understanding of the abnormal modification involved in carcinogenesis and progression. This review will provide a defined overview of these recent progression, with the aim of augmenting our knowledge on the role of PRMTs in progression and their potential application in cancer therapy.

OsPRMT6a-mediated arginine methylation of OsJAZ1 regulates jasmonate signaling and spikelet development in rice.

Although both protein arginine methylation (PRMT) and jasmonate (JA) signaling are crucial for regulating plant development, the relationship between these processes in the control of spikelet development remains unclear. In this study, we used the CRISPR/Cas9 technology to generate two OsPRMT6a loss-of-function mutants that exhibit various abnormal spikelet structures. Interestingly, we found that OsPRMT6a can methylate arginine residues in JA signal repressors OsJAZ1 and OsJAZ7. We showed that arginine methylation of OsJAZ1 enhances the binding affinity of OsJAZ1 with the JA receptors OsCOI1a and OsCOI1b in the presence of JAs, thereby promoting the ubiquitination of OsJAZ1 by the SCFOsCOI1a/OsCOI1b complex and degradation via the 26S proteasome. This process ultimately releases OsMYC2, a core transcriptional regulator in the JA signaling pathway, to activate or repress JA-responsive genes, thereby maintaining normal plant (spikelet) development. However, in the osprmt6a-1 mutant, reduced arginine methylation of OsJAZ1 impaires the interaction between OsJAZ1 and OsCOI1a/OsCOI1b in the presence of JAs. As a result, OsJAZ1 proteins become more stable, repressing JA responses, thus causing the formation of abnormal spikelet structures. Moreover, we discovered that JA signaling reduces the OsPRMT6a mRNA level in an OsMYC2-dependent manner, thereby establishing a negative feedback loop to balance JA signaling. We further found that OsPRMT6a-mediated arginine methylation of OsJAZ1 likely serves as a switch to tune JA signaling to maintain normal spikelet development under harsh environmental conditions such as high temperatures. Collectively, our study establishes a direct molecular link between arginine methylation and JA signaling in rice.

Research Progress on CARM1 and its Relationship with Colorectal Cancer.

Coactivator-associated arginine methyltransferase 1 (CARM1) is significant as a key member of the PRMT family, crucial for regulating arginine methylation, and its association with colorectal cancer underscores its potential as a therapeutic target. Consequently, CARM1 inhibitors have emerged as potential therapeutic agents in cancer treatment and valuable chemical tools for cancer research. Despite steady progress in CARM1 inhibitor research, challenges persist in discovering effective, isoform-selective, cell-permeable, and in vivo-active CARM1 inhibitors for colorectal cancer. This review summarizes the research progress on CARM1 and its relationship with colorectal cancer, aiming to provide a theoretical basis for the radiotherapy of colorectal cancer.

Characterization of the skeletal muscle arginine methylome in health and disease reveals remodeling in amyotrophic lateral sclerosis.

Arginine methylation is a protein posttranslational modification important for the development of skeletal muscle mass and function. Despite this, our understanding of the regulation of arginine methylation under settings of health and disease remains largely undefined. Here, we investigated the regulation of arginine methylation in skeletal muscles in response to exercise and hypertrophic growth, and in diseases involving metabolic dysfunction and atrophy. We report a limited regulation of arginine methylation under physiological settings that promote muscle health, such as during growth and acute exercise, nor in disease models of insulin resistance. In contrast, we saw a significant remodeling of asymmetric dimethylation in models of atrophy characterized by the loss of innervation, including in muscle biopsies from patients with myotrophic lateral sclerosis (ALS). Mass spectrometry-based quantification of the proteome and asymmetric arginine dimethylome of skeletal muscle from individuals with ALS revealed the largest compendium of protein changes with the identification of 793 regulated proteins, and novel site-specific changes in asymmetric dimethyl arginine (aDMA) of key sarcomeric and cytoskeletal proteins. Finally, we show that in vivo overexpression of PRMT1 and aDMA resulted in increased fatigue resistance and functional recovery in mice. Our study provides evidence for asymmetric dimethylation as a regulator of muscle pathophysiology and presents a valuable proteomics resource and rationale for numerous methylated and nonmethylated proteins, including PRMT1, to be pursued for therapeutic development in ALS.

The emerging role of CARM1 in cancer.

Coactivator-associated arginine methyltransferase 1 (CARM1), pivotal for catalyzing arginine methylation of histone and non-histone proteins, plays a crucial role in developing various cancers. CARM1 was initially recognized as a transcriptional coregulator by orchestrating chromatin remodeling, transcription regulation, mRNA splicing and stability. This diverse functionality contributes to the recruitment of transcription factors that foster malignancies. Going beyond its established involvement in transcriptional control, CARM1-mediated methylation influences a spectrum of biological processes, including the cell cycle, metabolism, autophagy, redox homeostasis, and inflammation. By manipulating these physiological functions, CARM1 becomes essential in critical processes such as tumorigenesis, metastasis, and therapeutic resistance. Consequently, it emerges as a viable target for therapeutic intervention and a possible biomarker for medication response in specific cancer types. This review provides a comprehensive exploration of the various physiological functions of CARM1 in the context of cancer. Furthermore, we discuss potential CARM1-targeting pharmaceutical interventions for cancer therapy.

Relationship between arginine methylation and vascular calcification.

In patients on maintenance hemodialysis (MHD), vascular calcification (VC) is an independent predictor of cardiovascular disease (CVD), which is the primary cause of death in chronic kidney disease (CKD). The main component of VC in CKD is the vascular smooth muscle cells (VSMCs). VC is an ordered, dynamic activity. Under the stresses of oxidative stress and calcium-­phosphorus imbalance, VSMCs undergo osteogenic phenotypic transdifferentiation, which promotes the formation of VC. In addition to traditional epigenetics like RNA and DNA control, post-translational modifications have been discovered to be involved in the regulation of VC in recent years. It has been reported that the process of osteoblast differentiation is impacted by catalytic histone or non-histone arginine methylation. Its function in the osteogenic process is comparable to that of VC. Thus, we propose that arginine methylation regulates VC via many signaling pathways, including as NF-B, WNT, AKT/PI3K, TGF-/BMP/SMAD, and IL-6/STAT3. It might also regulate the VC-related calcification regulatory factors, oxidative stress, and endoplasmic reticulum stress. Consequently, we propose that arginine methylation regulates the calcification of the arteries and outline the regulatory mechanisms involved.

Protein Arginine Methyltransferases in Pancreatic Ductal Adenocarcinoma: New Molecular Targets for Therapy.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignant disease with a low 5-year overall survival rate. It is the third-leading cause of cancer-related deaths in the United States. The lack of robust therapeutics, absence of effective biomarkers for early detection, and aggressive nature of the tumor contribute to the high mortality rate of PDAC. Notably, the outcomes of recent immunotherapy and targeted therapy against PDAC remain unsatisfactory, indicating the need for novel therapeutic strategies. One of the newly described molecular features of PDAC is the altered expression of protein arginine methyltransferases (PRMTs). PRMTs are a group of enzymes known to methylate arginine residues in both histone and non-histone proteins, thereby mediating cellular homeostasis in biological systems. Some of the PRMT enzymes are known to be overexpressed in PDAC that promotes tumor progression and chemo-resistance via regulating gene transcription, cellular metabolic processes, RNA metabolism, and epithelial mesenchymal transition (EMT). Small-molecule inhibitors of PRMTs are currently under clinical trials and can potentially become a new generation of anti-cancer drugs. This review aims to provide an overview of the current understanding of PRMTs in PDAC, focusing on their pathological roles and their potential as new therapeutic targets.

Deficiency of betaine-homocysteine methyltransferase activates glucose-6-phosphate dehydrogenase (G6PD) by decreasing arginine methylation of G6PD in hepatocellular carcinogenesis.

Betaine-homocysteine methyltransferase (BHMT) regulates protein methylation and is correlated with tumorigenesis; however, the effects and regulation of BHMT in hepatocarcinogenesis remain largely unexplored. Here, we determined the clinical significance of BHMT in the occurrence and progression of hepatocellular carcinoma (HCC) using tissue samples from 198 patients. BHMT was to be frequently found (86.6%) expressed at relatively low levels in HCC tissues and was positively correlated with the overall survival of patients with HCC. Bhmt overexpression effectively suppressed several malignant phenotypes in hepatoma cells in vitro and in vivo, whereas complete knockout of Bhmt (Bhmt-/-) produced the opposite effect. We combined proteomics, metabolomics, and molecular biological strategies and detected that Bhmt-/- promoted hepatocarcinogenesis and tumor progression by enhancing the activity of glucose-6-phosphate dehydrogenase (G6PD) and PPP metabolism in DEN-induced HCC mouse and subcutaneous tumor-bearing models. In contrast, restoration of Bhmt with an AAV8-Bhmt injection or pharmacological inhibition of G6PD attenuated hepatocarcinogenesis. Additionally, coimmunoprecipitation identified monomethylated modifications of the G6PD, and BHMT regulated the methylation of G6PD. Protein sequence analysis, generation and application of specific antibodies, and site-directed mutagenesis indicated G6PD methylation at the arginine residue 246. Furthermore, we established bidirectionally regulated BHMT cellular models combined with methylation-deficient G6PD mutants to demonstrate that BHMT potentiated arginine methylation of G6PD, thereby inhibiting G6PD activity, which in turn suppressed hepatocarcinogenesis. Taken together, this study reveals a new methylation-regulatory mechanism in hepatocarcinogenesis owing to BHMT deficiency, suggesting a potential therapeutic strategy for HCC treatment.

Arginine methylation and respiratory disease.

Arginine methylation, a vital post-translational modification, plays a pivotal role in numerous cellular functions such as signal transduction, DNA damage response and repair, regulation of gene transcription, mRNA splicing, and protein interactions. Central to this modification is the role of protein arginine methyltransferases (PRMTs), which have been increasingly recognized for their involvement in the pathogenesis of various respiratory diseases. This review begins with an exploration of the biochemical underpinnings of arginine methylation, shedding light on the intricate molecular regulatory mechanisms governed by PRMTs. It then delves into the impact of arginine methylation and the dysregulation of arginine methyltransferases in diverse pulmonary disorders. Concluding with a focus on the therapeutic potential and recent advancements in PRMT inhibitors, this article aims to offer novel perspectives and therapeutic avenues for the management and treatment of respiratory diseases.

PRMT5-mediated homologous recombination repair is essential to maintain genomic integrity of neural progenitor cells.

Maintaining genomic stability is a prerequisite for proliferating NPCs to ensure genetic fidelity. Though histone arginine methylation has been shown to play important roles in safeguarding genomic stability, the underlying mechanism during brain development is not fully understood. Protein arginine N-methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase that plays a role in transcriptional regulation. Here, we identify PRMT5 as a key regulator of DNA repair in response to double-strand breaks (DSBs) during NPC proliferation. Prmt5F/F; Emx1-Cre (cKO-Emx1) mice show a distinctive microcephaly phenotype, with partial loss of the dorsal medial cerebral cortex and complete loss of the corpus callosum and hippocampus. This phenotype is resulted from DSBs accumulation in the medial dorsal cortex followed by cell apoptosis. Both RNA sequencing and in vitro DNA repair analyses reveal that PRMT5 is required for DNA homologous recombination (HR) repair. PRMT5 specifically catalyzes H3R2me2s in proliferating NPCs in the developing mouse brain to enhance HR-related gene expression during DNA repair. Finally, overexpression of BRCA1 significantly rescues DSBs accumulation and cell apoptosis in PRMT5-deficient NSCs. Taken together, our results show that PRMT5 maintains genomic stability by regulating histone arginine methylation in proliferating NPCs.

Dynein Light Intermediate Chains Exhibit Different Arginine Methylation Patterns.

BACKGROUND: The motor protein dynein is integral to retrograde transport along microtubules and interacts with numerous cargoes through the recruitment of cargo-specific adaptor proteins. This interaction is mediated by dynein light intermediate chain subunits LIC1 (DYNC1LI1) and LIC2 (DYNC1LI2), which govern the adaptor binding and are present in distinct dynein complexes with overlapping and unique functions. METHODS: Using bioinformatics, we analyzed the C-terminal domains (CTDs) of LIC1 and LIC2, revealing similar structural features but diverse post-translational modifications (PTMs). The methylation status of LIC2 and the proteins involved in this modification were examined through immunoprecipitation and immunoblotting analyses. The specific methylation sites on LIC2 were identified through a site-directed mutagenesis analysis, contributing to a deeper understanding of the regulatory mechanisms of the dynein complex. RESULTS: We found that LIC2 is specifically methylated at the arginine 397 residue, a reaction that is catalyzed by protein arginine methyltransferase 1 (PRMT1). CONCLUSIONS: The distinct PTMs of the LIC subunits offer a versatile mechanism for dynein to transport diverse cargoes efficiently. Understanding how these PTMs influence the functions of LIC2, and how they differ from LIC1, is crucial for elucidating the role of dynein-related transport pathways in a range of diseases. The discovery of the arginine 397 methylation site on LIC2 enhances our insight into the regulatory PTMs of dynein functions.

Arginine methylation-dependent cGAS stability promotes non-small cell lung cancer cell proliferation.

Cyclic GMP-AMP synthase (cGAS), promotes non-small cell lung cancer (NSCLC) cell proliferation. However, the specific mechanisms of cGAS-mediated NSCLC cell proliferation are largely unknown. In this study, we found asymmetric dimethylation by protein arginine methyltransferase 1 (PRMT1) at R127 of cGAS. This facilitated the binding of deubiquitinase USP7 and contributed to deubiquitination and stabilization of cGAS. PRMT1-and USP7-dependent cGAS stability, which also played a pivotal role in accelerating NSCLC cell proliferation through activating AKT pathway. We validated that the expression of cGAS and PRMT1 were positive correlated in human non-small cell lung cancer samples. Our study demonstrates a unique mechanism for managing cGAS stability by arginine methylation and indicates that PRMT1-cGAS-USP7 axis is a potential therapeutic target for NSCLC.

Hypoxia-inducible PRMT2 addiction in glioblastomas.

Hypoxia-inducible transcription factors (HIFs) are key transcription factors for cellular response to low oxygen levels. However, the specific mediators responsible for activating downstream transcription are not well characterized. We previously identified Protein Arginine methyltransferase 2 (PRMT2), a highly expressed methyltransferase in glioblastoma multiforme, as a transcription co-activator. And we established a connection between PRMT2-mediated histone H3R8 asymmetric methylation (H3R8me2a) and transcription activation. Here we find that PRMT2 is activated by HIF1α under hypoxic conditions. And we demonstrate that PRMT2 and its H3R8me2a activity are required for the transcription activation of a significant subset of hypoxia-induced genes. Consequently, the inactivation of PRMT2 suppresses hypoxia-induced glioblastoma cell migration, attenuates tumor progression, and enhances chemotherapeutic sensitivity in mouse xenograft models. In addition, our analysis of clinical glioma specimens reveals a correlation between PRMT2 protein levels, HIF1α abundance, and an unfavorable prognosis. Our study establishes HIF1α-induced PRMT2 as a critical modulator in the activation of hypoxia-related transcriptional programs, ultimately driving malignant progression.

PRMT1 in human neoplasm: cancer biology and potential therapeutic target.

Protein arginine methyltransferase 1 (PRMT1), the predominant type I protein arginine methyltransferase, plays a crucial role in normal biological functions by catalyzing the methylation of arginine side chains, specifically monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), within proteins. Recent investigations have unveiled an association between dysregulated PRMT1 expression and the initiation and progression of tumors, significantly impacting patient prognosis, attributed to PRMT1's involvement in regulating various facets of tumor cell biology, including DNA damage repair, transcriptional and translational regulation, as well as signal transduction. In this review, we present an overview of recent advancements in PRMT1 research across different tumor types, with a specific focus on its contributions to tumor cell proliferation, metastasis, invasion, and drug resistance. Additionally, we expound on the dynamic functions of PRMT1 during distinct stages of cancer progression, elucidating its unique regulatory mechanisms within the same signaling pathway and distinguishing between its promotive and inhibitory effects. Importantly, we sought to provide a comprehensive summary and analysis of recent research progress on PRMT1 in tumors, contributing to a deeper understanding of its role in tumorigenesis, development, and potential treatment strategies.

Genetically encoded fluorescent sensor to monitor intracellular arginine methylation.

Arginine methylation (ArgMet), as a post-translational modification, plays crucial roles in RNA processing, transcriptional regulation, signal transduction, DNA repair, apoptosis and liquid-liquid phase separation (LLPS). Since arginine methylation is associated with cancer pathogenesis and progression, protein arginine methyltransferases have gained interest as targets for anti-cancer therapy. Despite considerable process made to elucidate (patho)physiological mechanisms regulated by arginine methylation, there remains a lack of tools to visualize arginine methylation with high spatiotemporal resolution in live cells. To address this unmet need, we generated an ArgMet-sensitive genetically encoded, Förster resonance energy transfer-(FRET) based biosensor, called GEMS, capable of quantitative real-time monitoring of ArgMet dynamics. We optimized these biosensors by using different ArgMet-binding domains, arginine-glycine-rich regions and adjusting the linkers within the biosensors to improve their performance. Using a set of mammalian cell lines and modulators, we demonstrated the applicability of GEMS for monitoring changes in arginine methylation with single-cell and temporal resolution. The GEMS can facilitate the in vitro screening to find potential protein arginine methyltransferase inhibitors and will contribute to a better understanding of the regulation of ArgMet related to differentiation, development and disease.

Integration of bulk RNA sequencing to reveal protein arginine methylation regulators have a good prognostic value in immunotherapy to treat lung adenocarcinoma.

Background: Given the differential expression and biological functions of protein arginine methylation (PAM) regulators in lung adenocarcinoma (LUAD), it may be of great value in the diagnosis, prognosis, and treatment of LUAD. However, the expression and function of PAM regulators in LUAD and its relationship with prognosis are unclear. Methods: 8 datasets including 1798 LUAD patients were selected. During the bioinformatic study in LUAD, we performed (i) consensus clustering to identify clusters based on 9 PAM regulators related expression profile data, (ii) to identify hub genes between the 2 clusters, (iii) principal component analysis to construct a PAM.score based on above genes, and (iv) evaluation of the effect of PAM.score on the deconstruction of tumor microenvironment and guidance of immunotherapy. Results: We identified two different clusters and a robust and clinically practicable prognostic scoring system. Meanwhile, a higher PAM.score subgroup showed poorer prognosis, and was validated by multiple cohorts. Its prognostic effect was validated by ROC (Receiver operating characteristic curve) curve and found to have a relatively good prediction efficacy. High PAM.score group exhibited lower immune score, which associated with an immunosuppressive microenvironment in LUAD. Finally, patients exhibiting a lower PAM.score presented noteworthy therapeutic benefits and clinical advantages. Conclusion: Our PAM.score model can help clinicians to select personalized therapy for LUAD patients, and PAM.score may act a part in the development of LUAD.

Protein Arginine Methylation Patterns in Plasma Small Extracellular Vesicles Are Altered in Patients with Early-Stage Pancreatic Ductal Adenocarcinoma.

Small extracellular vesicles (sEVs) contain lipids, proteins and nucleic acids, which often resemble their cells of origin. Therefore, plasma sEVs are considered valuable resources for cancer biomarker development. However, previous efforts have been largely focused on the level of proteins and miRNAs in plasma sEVs, and the post-translational modifications of sEV proteins, such as arginine methylation, have not been explored. Protein arginine methylation, a relatively stable post-translational modification, is a newly described molecular feature of PDAC. The present study examined arginine methylation patterns in plasma sEVs derived from patients with early-stage PDAC (n = 23) and matched controls. By utilizing the arginine methylation-specific antibodies for western blotting, we found that protein arginine methylation patterns in plasma sEVs are altered in patients with early-stage PDAC. Specifically, we observed a reduction in the level of symmetric dimethyl arginine (SDMA) in plasma sEV proteins derived from patients with early- and late-stage PDAC. Importantly, immunoprecipitation followed by proteomics analysis identified a number of arginine-methylated proteins exclusively present in plasma sEVs derived from patients with early-stage PDAC. These results indicate that arginine methylation patterns in plasma sEVs are potential indicators of PDAC, a new concept meriting further investigation.

Arginine-methylated c-Myc affects mitochondrial mitophagy in mouse acute kidney injury via Slc25a24.

The transcription factor methylated c-Myc heterodimerizes with MAX to modulate gene expression, and plays an important role in energy metabolism in kidney injury but the exact mechanism remains unclear. Mitochondrial solute transporter Slc25a24 imports ATP into mitochondria and is central to energy metabolism. Gene Expression Omnibus data analysis reveals Slc25a24 and c-Myc are consistently upregulated in all the acute kidney injury (AKI) cells. Pearson correlation analysis also shows that Slc25a24 and c-Myc are strongly correlated (⍴ > 0.9). Mutant arginine methylated c-Myc (R299A and R346A) reduced its combination with MAX when compared with the wild type of c-Myc. On the other hand, the Slc25a24 levels were also correspondingly reduced, which induced the downregulation of ATP production. The results promoted reactive oxygen species (ROS) production and mitophagy generation. The study revealed that the c-Myc overexpression manifested the most pronounced mitochondrial DNA depletion. Additionally, the varied levels of mitochondrial proteins like TIM23, TOM20, and PINK1 in each group, particularly the elevated levels of PINK1 in AKI model groups and lower levels of TIM23 and TOM20 in the c-Myc overexpression group, suggest potential disruptions in mitochondrial dynamics and homeostasis, indicating enhanced mitophagy or mitochondrial loss. Therefore, arginine-methylated c-Myc affects mouse kidney injury by regulating mitochondrial ATP and ROS, and mitophagy via Slc25a24.

Protein arginine methyltransferase 1 is required for the maintenance of adult small intestinal and colonic epithelial cell homeostasis.

The vertebrate adult intestinal epithelium has a high self-renewal rate driven by intestinal stem cells (ISCs) in the crypts, which play central roles in maintaining intestinal integrity and homeostasis. However, the underlying mechanisms remain elusive. Here we showed that protein arginine methyltransferase 1 (PRMT1), a major arginine methyltransferase that can also function as a transcription co-activator, was highly expressed in the proliferating cells of adult mouse intestinal crypts. Intestinal epithelium-specific knockout of PRMT1, which ablates PRMT1 gene starting during embryogenesis, caused distinct, region-specific effects on small intestine and colon: increasing and decreasing the goblet cell number in the small intestinal and colonic crypts, respectively, leading to elongation of the crypts in small intestine but not colon, while increasing crypt cell proliferation in both regions. We further generated a tamoxifen-inducible intestinal epithelium-specific PRMT1 knockout mouse model and found that tamoxifen-induced knockout of PRMT1 in the adult mice resulted in the same region-specific intestinal phenotypes. Thus, our studies have for the first time revealed that the epigenetic enzyme PRMT1 has distinct, region-specific roles in the maintenance of intestinal epithelial architecture and homeostasis, although PRMT1 may influence intestinal development.