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Steven Clarke - One of the best experts on this subject based on the ideXlab platform.
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a glutamate aspartate switch controls product specificity in a Protein Arginine Methyltransferase
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Erik W Debler, Steven Clarke, You Feng, Kanishk Jain, Rebeccah A Warmack, Gunter Blobel, Pete StavropoulosAbstract:Trypanosoma brucei PRMT7 (TbPRMT7) is a Protein Arginine Methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 A resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved Methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, Methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target Arginine in substrate peptides/Proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylArginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of Protein Arginine Methyltransferases and have important implications for the rational (re)design of PRMTs.
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A glutamate/aspartate switch controls product specificity in a Protein Arginine Methyltransferase
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Erik W Debler, Steven Clarke, You Feng, Kanishk Jain, Rebeccah A Warmack, Gunter Blobel, Pete StavropoulosAbstract:Trypanosoma brucei PRMT7 (TbPRMT7) is a Protein Arginine Methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 A resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved Methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, Methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target Arginine in substrate peptides/Proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylArginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of Protein Arginine Methyltransferases and have important implications for the rational (re)design of PRMTs.
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unique features of human Protein Arginine Methyltransferase 9 prmt9 and its substrate rna splicing factor sf3b2
Journal of Biological Chemistry, 2015Co-Authors: Andrea Hadjikyriacou, Yanzhong Yang, Alexsandra Espejo, Mark T Bedford, Steven ClarkeAbstract:Human Protein Arginine Methyltransferase (PRMT) 9 symmetrically dimethylates Arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated Methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. Although amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the Arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylArginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated Methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides.
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substrate specificity of human Protein Arginine Methyltransferase 7 prmt7 the importance of acidic residues in the double e loop
Journal of Biological Chemistry, 2014Co-Authors: You Feng, Andrea Hadjikyriacou, Steven ClarkeAbstract:Protein Arginine Methyltransferase 7 (PRMT7) methylates Arginine residues on various Protein substrates and is involved in DNA transcription, RNA splicing, DNA repair, cell differentiation, and metastasis. The substrate sequences it recognizes in vivo and the enzymatic mechanism behind it, however, remain to be explored. Here we characterize methylation catalyzed by a bacterially expressed GST-tagged human PRMT7 fusion Protein with a broad range of peptide and Protein substrates. After confirming its type III activity generating only ω-NG-monomethylArginine and its distinct substrate specificity for RXR motifs surrounded by basic residues, we performed site-directed mutagenesis studies on this enzyme, revealing that two acidic residues within the double E loop, Asp-147 and Glu-149, modulate the substrate preference. Furthermore, altering a single acidic residue, Glu-478, on the C-terminal domain to glutamine nearly abolished the activity of the enzyme. Additionally, we demonstrate that PRMT7 has unusual temperature dependence and salt tolerance. These results provide a biochemical foundation to understanding the broad biological functions of PRMT7 in health and disease.
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Hsl7 is a substrate-specific type II Protein Arginine Methyltransferase in yeast.
Biochemical and biophysical research communications, 2008Co-Authors: Joyce Sayegh, Steven ClarkeAbstract:The Saccharomyces cerevisiae Protein Hsl7 is a regulator of the Swe1 Protein kinase in cell cycle checkpoint control. Hsl7 has been previously described as a type III Protein Arginine Methyltransferase, catalyzing the formation of omega-monomethylArginine residues on non-physiological substrates. However, we show here that Hsl7 can also display type II activity, generating symmetric dimethylArginine residues on calf thymus histone H2A. Symmetric dimethylation is only observed when enzyme and the methyl-accepting substrate were incubated for extended times. We confirmed the Hsl7-dependent formation of symmetric dimethylArginine by amino acid analysis and thin layer chromatography with wild-type and mutant recombinant enzymes expressed from both bacteria and yeast. This result is significant because no type II activity has been previously demonstrated in S. cerevisiae. We also show that Hsl7 has little or no activity on GST-GAR, a commonly used substrate for Protein Arginine Methyltransferases, and only minimal activity on myelin basic Protein. This enzyme thus may only recognize only a small subset of potential substrate Proteins in yeast, in contrast to the situation with Rmt1, the major type I Methyltransferase.
Mark T Bedford - One of the best experts on this subject based on the ideXlab platform.
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unique features of human Protein Arginine Methyltransferase 9 prmt9 and its substrate rna splicing factor sf3b2
Journal of Biological Chemistry, 2015Co-Authors: Andrea Hadjikyriacou, Yanzhong Yang, Alexsandra Espejo, Mark T Bedford, Steven ClarkeAbstract:Human Protein Arginine Methyltransferase (PRMT) 9 symmetrically dimethylates Arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated Methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. Although amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the Arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylArginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated Methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides.
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Mammalian Protein Arginine Methyltransferase 7 (PRMT7) specifically targets RXR sites in lysine- and Arginine-rich regions.
The Journal of biological chemistry, 2013Co-Authors: You Feng, Andrea Hadjikyriacou, Mark T Bedford, Ranjan Maity, Julian P. Whitelegge, Cecilia Zurita-lopez, Qais Al-hadid, Amander T. Clark, Jean-yves MassonAbstract:The mammalian Protein Arginine Methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust Methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylArginine residues, and we confirm its activity as the prototype type III Protein Arginine Methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating Arginine residues in lysine- and Arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of Arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7.
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methylation by Protein Arginine Methyltransferase 1 increases stability of axin a negative regulator of wnt signaling
Oncogene, 2011Co-Authors: B Cha, Mark T Bedford, Won Ho Kim, Yong Kee Kim, B N Hwang, Sun Park, J W Yoon, Won Sang Park, Jin Won Cho, Eekhoon JhoAbstract:Axin, a negative regulator of Wnt signaling, is a key scaffold Protein for the β-catenin destruction complex. It has been previously shown that multiple post-translational modification enzymes regulate the level of Axin. Here, we provide evidence that Protein Arginine Methyltransferase 1 (PRMT1) directly interacts with and methylates the 378th Arginine residue of Axin both in vitro and in vivo. We found that the transient expression of PRMT1 led to an increased level of Axin and that knockdown of endogenous PRMT1 by short hairpin RNA reduced the level of Axin. These results suggest that methylation by PRMT1 enhanced the stability of Axin. Methylation of Axin by PRMT1 also seemingly enhanced the interaction between Axin and glycogen synthase kinase 3β, leading to decreased ubiquitination of Axin. Consistent with the role of PRMT1 in the regulation of Axin, knockdown of PRMT1 enhanced the level of cytoplasmic β-catenin as well as β-catenin-dependent transcription activity. In summary, we show that the methylation of Axin occurred in vivo and controlled the stability of Axin. Therefore, methylation of Axin by PRMT1 may serve as a finely tuned regulation mechanism for Wnt/β-catenin signaling.
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epigenetic regulation of transcriptional activity of pregnane x receptor by Protein Arginine Methyltransferase 1
Journal of Biological Chemistry, 2009Co-Authors: Ying Xie, Mark T Bedford, Nengtai Ouyang, Wen Xie, Yanan TianAbstract:Pregnane X receptor (PXR) is a ligand-dependent transcription factor, regulating gene expression of enzymes and transporters involved in xenobiotic/drug metabolism. Here, we report that Protein Arginine Methyltransferase 1 (PRMT1) is required for the transcriptional activity of PXR. PRMT1 regulates expression of numerous genes, including nuclear receptor-regulated transcription, through methylating histone and non-histone Proteins. Co-immunoprecipitation and histone Methyltransferase assays revealed that PRMT1 is a major histone Methyltransferase associated with PXR. The PXR ligand-binding domain is responsible for PXR-PRMT1 interaction as determined by mammalian two-hybrid and glutathione S-transferase (GST) pull-down assays. The chromatin immunoprecipitation (ChIP) assay showed that PRMT1 was recruited to the regulatory region of the PXR target gene cytochrome P450 3A4 (CYP3A4), with a concomitant methylation of Arginine 3 of histone H4, in response to the PXR agonist rifampicin. In mammalian cells, small interfering RNA (siRNA) knockdown and gene deletion of PRMT1 greatly diminished the transcriptional activity of PXR, suggesting an indispensable role of PRMT1 in PXR-regulated gene expression. Interestingly, PXR appears to have a reciprocal effect on the PRMT1 functions by regulating its cellular compartmentalization as well as its substrate specificity. Taken together, these results demonstrated mutual interactions and functional interplays between PXR and PRMT1, and this interaction may be important for the epigenetics of PXR-regulated gene expression.
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Protein Arginine Methyltransferase 1 coactivates nf κb dependent gene expression synergistically with carm1 and parp1
Journal of Molecular Biology, 2008Co-Authors: Paul O Hassa, Mark T Bedford, Marcela Covic, Michael O HottigerAbstract:Nuclear factor kappa B (NF-kappaB) plays an important role in the transcriptional regulation of genes involved in inflammation and cell survival. Transcriptional coactivators that methylate histones become increasingly important. Recently, we provided evidence that coactivator-associated Arginine Methyltransferase 1 (CARM1) is a transcriptional coactivator of NF-kappaB and functions as a promoter-specific regulator of NF-kappaB recruitment to chromatin. Here, we show that Protein Arginine Methyltransferase 1 (PRMT1) synergistically coactivates NF-kappaB-dependent gene expression at the macrophage inflammatory Protein 2 and human immunodeficiency virus 1 long terminal repeat promoters in concert with the transcriptional coactivators p300/CREB binding Protein, CARM1, and poly(ADP-ribose) polymerase 1. PRMT1 formed a complex with poly(ADP-ribose) polymerase 1 and NF-kappaB in vivo and interacted directly with the NF-kappaB subunit p65 in vitro. The Methyltransferase activity of PRMT1 appeared essential for its coactivator function in context with CARM1 and p300/CREB binding Protein. These results suggest that the cooperative action between PRMT1 and CARM1 is required for NF-kappaB-dependent gene expression.
Vladimir Ljubicic - One of the best experts on this subject based on the ideXlab platform.
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Protein Arginine Methyltransferase biology in humans during acute and chronic skeletal muscle plasticity
Journal of applied physiology (Bethesda Md. : 1985), 2019Co-Authors: Tiffany L. Vanlieshout, Jacob T. Bonafiglia, Brendon J. Gurd, Vladimir LjubicicAbstract:This is the first report of Protein Arginine Methyltransferase (PRMT) biology in human skeletal muscle in vivo. We observed that PRMT1, -4, -5, and -7 were the most abundant PRMT mRNAs in human mus...
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Protein Arginine Methyltransferase Expression During An Acute Time Course Of Denervation-Induced Skeletal Muscle Plasticity
The FASEB Journal, 2016Co-Authors: Derek W Stouth, Vladimir LjubicicAbstract:Protein Arginine Methyltransferase 1 (PRMT1) and co-activator-associated Arginine Methyltransferase 1 (CARM1) are critical components of a diverse set of intracellular functions including cell sign...
William J. Sullivan - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Protein Arginine Methyltransferase of TgPRMT5 in Toxoplasma gondii
Parasites & vectors, 2019Co-Authors: Min Liu, Ting-kai Liu, Long-fei Chen, Pei-liang Yang, Zhi-fa Lai, William J. SullivanAbstract:Background Protein Arginine methylation is a prevalent post-translational modification. The Protein Arginine Methyltransferase family (PRMT) is involved in many cellular processes in eukaryotes, including transcriptional regulation, epigenetic regulation, RNA metabolism, and DNA damage repair. Toxoplasma gondii, an opportunistic protozoan parasite, encodes five conserved PRMTs. PRMT5 is thought to be responsible for substantial PRMT activity in T. gondii; however, it has not yet been characterized.
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Characterization of Protein Arginine Methyltransferase of TgPRMT5 in Toxoplasma gondii
BMC, 2019Co-Authors: Min Liu, Ting-kai Liu, Long-fei Chen, Pei-liang Yang, Zhi-fa Lai, William J. SullivanAbstract:Abstract Background Protein Arginine methylation is a prevalent post-translational modification. The Protein Arginine Methyltransferase family (PRMT) is involved in many cellular processes in eukaryotes, including transcriptional regulation, epigenetic regulation, RNA metabolism, and DNA damage repair. Toxoplasma gondii, an opportunistic protozoan parasite, encodes five conserved PRMTs. PRMT5 is thought to be responsible for substantial PRMT activity in T. gondii; however, it has not yet been characterized. Methods We tagged the 3′ end of the endogenous TgPRMT5 genomic locus with sequence encoding a 3X hemagglutinin (HA) epitope. IFA and WB were performed to check the expression and subcellular localization of TgPRMT5 in tachyzoites and bradyzoites. In vitro methylation assays were performed to determine whether endogenous TgPRMT5 has Arginine Methyltransferase activity. Results IFA and WB results showed that T. gondii PRMT5 (TgPRMT5) was localized in the cytoplasm in the tachyzoite stage; however, it shifts largely to the nuclear compartment in the bradyzoite stage. The in vitro methylation showed that TgPRMT5 has authentic type II PRMT activity and forms monomethylArginines and symmetric dimethylArginines. Conclusions We determined the expression and cellular localization of TgPRMT5 in tachyzoites and bradyzoites and confirmed its type II PRMT activity. We demonstrated the major changes in expression and cellular localization of TgPRMT5 during the tachyzoite and bradyzoite stages in T. gondii. Our findings suggest that TgPRMT5 Protein may be involved in tachyzoite-bradyzoite transformation
Didier Meseure - One of the best experts on this subject based on the ideXlab platform.
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Protein Arginine Methyltransferase 5: A novel therapeutic target for triple-negative breast cancers
Cancer Medicine, 2019Co-Authors: Mathilde Vinet, Amélie Brisson, Virginie Maire, Samyuktha Suresh, Clarisse Monchecourt, Fariba Nemati, Laëtitia Lesage, Fabienne Pierre, Auriane Lescure, Didier MeseureAbstract:TNBC is a highly heterogeneous and aggressive breast cancer subtype associated with high relapse rates, and for which no targeted therapy yet exists. Protein Arginine Methyltransferase 5 (PRMT5), an enzyme which catalyzes the methylation of Arginines on histone and non-histone Proteins, has recently emerged as a putative target for cancer therapy. Potent and specific PRMT5 inhibitors have been developed, but the therapeutic efficacy of PRMT5 targeting in TNBC has not yet been demonstrated. Here, we examine the expression of PRMT5 in a human breast cancer cohort obtained from the Institut Curie, and evaluate the therapeutic potential of pharmacological inhibition of PRMT5 in TNBC. We find that PRMT5 mRNA and Protein are expressed at comparable levels in TNBC, luminal breast tumors, and healthy mammary tissues. However, immunohistochemistry analyses reveal that PRMT5 is differentially localized in TNBC compared to other breast cancer subtypes and to normal breast tissues. PRMT5 is heterogeneously expressed in TNBC and high PRMT5 expression correlates with poor prognosis within this breast cancer subtype. Using the small-molecule inhibitor EPZ015666, we show that PRMT5 inhibition impairs cell proliferation in a subset of TNBC cell lines. PRMT5 inhibition triggers apoptosis, regulates cell cycle progression and decreases mammosphere formation. Furthermore, EPZ015666 administration to a patient-derived xenograft model of TNBC significantly deters tumor progression. Finally, we reveal potentiation between EGFR and PRMT5 targeting, suggestive of a beneficial combination therapy. Our findings highlight a distinctive subcellular localization of PRMT5 in TNBC, and uphold PRMT5 targeting, alone or in combination, as a relevant treatment strategy for a subset of TNBC.
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Abstract 3809: Protein Arginine Methyltransferase 1 (PRMT1) is a candidate therapeutic target for breast cancers
Experimental and Molecular Therapeutics, 2016Co-Authors: David C. Silvestre, Amélie Brisson, Bérengère Marty-prouvost, Hélène Bonsang, Virginie Maire, Damarys Loew, David Gentien, Didier Meseure, Fabien ReyalAbstract:Triple-negative breast cancer (TNBC) represents a subgroup of breast cancers associated with the most aggressive clinical behavior. No targeted therapy is currently available for the treatment of patients with TNBC. In the present study, we found that Protein Arginine Methyltransferase 1 (PRMT1) is overexpressed in TNBC at the mRNA level. At the Protein level, PRMT1 is overexpressed in all breast cancer subtypes compared to normal breast tissues. The depletion of PRMT1 using siRNA in breast cancer cell lines triggered apoptosis, reduced cell viability and the ability to form colonies in an anchorage-independent manner. Treatment with a PRMT1 inhibitor blocked proliferation specifically in breast cancer cells, with no effect in normal breast cells. Importantly, the expression of PRMT1 is an indicator of prognosis and response to treatment specifically in TNBC patients. To address the cellular pathways regulated by PRMT1, we identified its Protein partners by mass spectrometry and the transcriptomic changes following its depletion in TNBC cell lines. Interestingly, we found that PRMT1 directly activates key oncogenic pathways. Furthermore, we found a synergistic interaction between PRMT1 inhibitors and inhibitors for some of those pathways. We show that PRMT1 activity is necessary for breast cancer cell survival and oncogenic pathway activation. Altogether, our results point out PRMT1 as an emerging target for the treatment of breast cancers. Citation Format: David Silvestre, Amelie Brisson, Berengere Marty-Prouvost, Mengliang Ye, Helene Bonsang, Virginie Maire, Damarys Loew, David Gentien, Didier Meseure, Fabien Reyal, Gordon C. Tucker, Sergio Roman-Roman, Thierry Dubois. Protein Arginine Methyltransferase 1 (PRMT1) is a candidate therapeutic target for breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3809.
