Biochemical Analysis of Arginine Methylation in Transcription

Tini, Marc; Naeem, Hina; Torchia, Joseph

doi:10.1007/978-1-59745-190-1_16

Cited by 10 publications

(10 citation statements)

References 20 publications

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“…In vitro methylation assay using bacterially expressed GST–G3BP1 was performed as described earlier (Tini et al, 2009). Briefly, F9 cells were transiently transfected with HA–PRMT1 (6 μg) in 100 mm culture dishes.…”

Section: Methodsmentioning

confidence: 99%

Arginine methylation of G3BP1 in response to Wnt3a regulates β-catenin mRNA

Bikkavilli

Malbon

2011

Journal of Cell Science

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Wnt/β-catenin signaling is essential for normal mammalian development. Wnt3a activates the Wnt/β-catenin pathway through stabilization of β-catenin; a process in which the phosphoprotein Dishevelled figures prominently. Protein arginine methylation in signaling complexes containing Dishevelled was investigated. Mass spectrometry of a prominent arginine-methylated, Dishevelled-associated protein identified the Ras GTPase activating protein-binding protein 1 G3BP1. Stimulation of totipotent mouse embryonic F9 cells with Wnt3a provoked increased methylation of G3BP1. We show that G3BP1 is a novel Ctnnb1 mRNA binding protein. Methylation of G3BP1 constitutes a molecular switch that regulates Ctnnb1 mRNA in response to Wnt3a. Thus, the protein arginine methylation that targets G3BP1 acts as a novel regulator of Ctnnb1 mRNA.

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Section: Methodsmentioning

confidence: 99%

Arginine methylation of G3BP1 in response to Wnt3a regulates β-catenin mRNA

Bikkavilli

Malbon

2011

Journal of Cell Science

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“…In vitro methylation assay, which evaluates the abilities of purified PRMTs to catalyze methylation of their substrates, is a well-accepted assay for studying arginine methylation [10][11][12] . The overall success of this assay largely depends upon the activity of the purified PRMTs.…”

Section: Introductionmentioning

confidence: 99%

“…The overall success of this assay largely depends upon the activity of the purified PRMTs. PRMTs can be expressed and purified from bacteria, or mammalian cells 10,11 . This in vitro methylation assay, as detailed in the protocol section, is based upon a method originally described by Tini and colleagues 10 .…”

Section: Introductionmentioning

confidence: 99%

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<em>In vitro </em>Methylation Assay to Study Protein Arginine Methylation

Bikkavilli

Avasarala

Scoyk

et al. 2014

JoVE

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Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3 H] methionine). Here we describe a stepby-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of Sadenosyl-L-[methyl-3 H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation. Video LinkThe video component of this article can be found at

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“…HATs and HDACs2 have balancing actions for histones acetylation. Methylation of histones6 is controlled by histones methyltransferases (HMTs) and histones demethylases (HDMs), while histones arginine methylation is catalyzed by the protein arginine N-methyltransferases family of enzymes 7–9. One, two, or three methylations are possible, with impact on gene expression/repression 10.…”

Section: Introductionmentioning

confidence: 99%

Interpreting clinical assays for histone deacetylase inhibitors

Martinet¹,

Bertrand²

2011

CMR

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As opposed to genetics, dealing with gene expressions by direct DNA sequence modifications, the term epigenetics applies to all the external influences that target the chromatin structure of cells with impact on gene expression unrelated to the sequence coding of DNA itself. In normal cells, epigenetics modulates gene expression through all development steps. When “imprinted” early by the environment, epigenetic changes influence the organism at an early stage and can be transmitted to the progeny. Together with DNA sequence alterations, DNA aberrant cytosine methylation and microRNA deregulation, epigenetic modifications participate in the malignant transformation of cells. Their reversible nature has led to the emergence of the promising field of epigenetic therapy. The efforts made to inhibit in particular the epigenetic enzyme family called histone deacetylases (HDACs) are described. HDAC inhibitors (HDACi) have been proposed as a viable clinical therapeutic approach for the treatment of leukemia and solid tumors, but also to a lesser degree for noncancerous diseases. Three epigenetic drugs are already arriving at the patient’s bedside, and more than 100 clinical assays for HDACi are registered on the National Cancer Institute website. They explore the eventual additive benefits of combined therapies. In the context of the pleiotropic effects of HDAC isoforms, more specific HDACi and more informative screening tests are being developed for the benefit of the patients.

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Biochemical Analysis of Arginine Methylation in Transcription

Cited by 10 publications

References 20 publications

Arginine methylation of G3BP1 in response to Wnt3a regulates β-catenin mRNA

Arginine methylation of G3BP1 in response to Wnt3a regulates β-catenin mRNA

<em>In vitro </em>Methylation Assay to Study Protein Arginine Methylation

Interpreting clinical assays for histone deacetylase inhibitors

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