This study was supported by a Merit Award from the U.S. Department of Veterans Affairs, and an NIH grant R01CA138528 to RW.

1. Preparation of Expression Constructs
<ol>
	<li>Before starting the protocol, clone PRMTs into mammalian expression vectors with an N-terminal epitope tag (Myc or HA), and the substrate in-frame with glutathione-S-transferase (GST) for high-level protein expression and purification from bacterial cell lysates, using standard molecular biological techniques.</li>
</ol>
2. Purification of PRMTs
<ol>
	<li>Use 100 mm culture dishes to maintain human bronchial epithelial cells (Beas2B). Passage the cells every 3 days and do not let them grow to confluence.</li>
	<li>On the day before transfection, seed 7.5 x 105 cells in 10 ml culture medium in a 100 mm culture dish. Swirl to evenly disperse the cells, and incubate at 37 &#176;C in a 5% humidified CO2 incubator.</li>
	<li>The next day, aspirate the media and add 5 ml of culture media without serum and antibiotics. Swirl and aspirate the medium. Repeat this step one more time, and finally add 5 ml of serum/antibiotic free medium and proceed to transfection.</li>
	<li>Dilute 6 &#956;g of plasmid DNA into 750 &#956;l of serum free medium in a sterile 1.5 ml plastic centrifuge tube.</li>
	<li>In another 1.5 ml plastic centrifuge tube, dilute 30 &#956;l of transfection reagent into 750 &#956;l of serum free medium.</li>
	<li>Transfer the diluted transfection reagent solution (step 2.5) to the tube containing diluted DNA (step 2.4). Mix gently by pipetting up and down several times.</li>
	<li>Incubate the transfection mixture at room temperature for 15 min.</li>
	<li>After 15 min, pipette the transfection mixture gently onto the cells, swirl to mix evenly, and incubate at 37 &#176;C in a 5% humidified CO2 incubator.</li>
	<li>After 3 hr, aspirate the medium containing transfection mixtures from the cells. Add fresh cell culture medium supplemented with serum and antibiotics.</li>
	<li>Incubate at 37 &#176;C in a 5% humidified CO2 incubator for 48 hr.</li>
	<li>After 48 hr, lyse the cells in 1 ml of lysis buffer (1x phosphate buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 1 &#181;g/ml phenlymethyl sulphonyl fluoride). Collect the lysates into a plastic centrifuge tube with a cell lifter and incubate the lysates on ice for 30 min.</li>
	<li>Centrifuge the lysates at 15,000 x g for 10 min on a bench-top centrifuge to clear the lysates from cell debris. [* It is highly recommended to confirm the expression of HA-PRMT1 via immunoblotting of an aliquot of the lysates with anti-HA antibodies, before proceeding to purification of the enzymes for methylation assay.]</li>
	<li>For purification of PRMTs, incubate 3 mg of cell lysates with 30 &#956;l of 50:50 suspension of anti-HA affinity matrix in phosphate buffered saline (PBS), and rotate overnight at 4 &#176;C in a tube rotator.</li>
	<li>Next day, spin the tubes at 14,000 x g on a bench-top centrifuge for 2 min to collect the beads. Discard the supernatant and wash the beads 3x&#160;with 1 ml of RIPA buffer [20 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA and 1% Triton-X-100] in each wash followed by centrifugation at 14,000 x g for 2 min. After the third wash, wash the beads once with 100 &#956;l of methylation buffer (50 mM Tris pH 8.5, 20 mM KCl, 10 mM MgCl2, 1 mM &#946;-mercaptoethanol, 100 mM sucrose). After the centrifugation, remove the supernatant. The beads containing the immobilized PRMTs are ready to be used in the methylation assay. [* It is highly recommended to use the immobilized PRMTs immediately in the methylation assay.]</li>
</ol>
3. Purification of Substrate
<ol>
	<li>Inoculate a single colony of GST-G3BP1 transformed BL21 into 5 ml of Luria-Bertani (LB) medium containing 100 &#956;g/ml ampicillin. Grow the cultures overnight at 37 &#176;C with shaking at 250 rpm.</li>
	<li>Next day, transfer the overnight culture to 50 ml of fresh LB medium and continue to grow to an OD600 of 0.5-0.7 (It takes about 2 hr).</li>
	<li>When the OD600 reaches 0.5-0.7, add isopropyl-&#946;-D-thiogalactoside (IPTG) to a final concentration of 1 mM and continue to culture for additional 4 hr at 37 &#176;C with shaking at 250 rpm.</li>
	<li>Harvest the cells by centrifugation at 1,500 x g for 10 min, and process the pellet for GST purification or store at -80 &#176;C.</li>
	<li>Re-suspend the bacterial pellet in 5 ml of lysis buffer (1x PBS with protease inhibitors, 100 &#956;g/ml of lysozyme, and 0.1% Triton-X-100) and incubate the lysates at 37 &#176;C for 30 min. At this time the lysates will appear turbid and viscous due to the release of bacterial genomic DNA.</li>
	<li>Shear the genomic DNA by sonication (10 pulses of 1 sec each with an amplitude of 30%).</li>
	<li>Clear the lysates by centrifugation at 15,000 x g for 10 min and transfer the clear supernatant to a 15 ml tube. If the supernatant is not clear repeat the centrifugation process one more time.</li>
	<li>Meanwhile, wash the GST-sepharose beads twice with ice cold PBS and make a 50:50 suspension of GST-sepharose in PBS. Add 200 &#956;l of the GST-sepharose suspension to the cleared bacterial cell lysate and rotate at 4 &#176;C for 1 hr.</li>
	<li>After 1 hr, centrifuge the tubes at 500 x g for 3 min to collect the beads. Wash the beads 3x&#160;with ice cold PBS.</li>
	<li>After the final wash, resuspend the beads in 100 &#956;l of elution buffer (50 mM Tris pH 8.0, 150 mM NaCl, 30 mM glutathione) and rotate at 4 &#176;C for 10 min.</li>
	<li>Centrifuge the tubes at 15,000 x g for 2 min and transfer the supernatant containing the purified protein into another plastic centrifuge tube. Carry out SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis on 5 &#956;l of the purified protein along with known amounts of bovine serum albumin (BSA) to estimate the concentration of the purified protein.</li>
</ol>
4. Methylation Assay
NOTE: Perform following steps in a separate area designated for radioisotope work and in compliance with the institution&#8217;s protocols on performing experiments with radioactive isotopes.
<ol>
	<li>Add 2 &#956;g of purified GST-substrate and a master mix containing 1x methylation buffer (50 mM Tris pH 8.5, 20 mM KCl, 10 mM MgCl2, 1 mM &#946;-mercaptoethanol, 100 mM sucrose), 1 &#956;Ci of S-adenosyl-L-[methyl-3H] methionine, and water to a final volume of 10 &#956;l to the immobilized PRMTs (step 2.13) and incubate the reaction at 30 &#176;C for 1 hr.</li>
	<li>After 1 hr, stop the reaction by adding 10 &#956;l of 2x SDS sample buffer. After denaturation of the samples at 95 &#176;C for 10 min, separate the samples on an SDS-PAGE gel.</li>
	<li>Later, transfer the separated proteins from the SDS gel onto nitrocellulose membrane by using a semi-dry electrophoretic transfer.</li>
	<li>After the transfer, cover the membrane with autofluor (an autoradiographic image intensifier), and gently rock for 2 hr at room temperature. After 2 hr, pour off the autofluor and retain for later use. [*Label the tube as radioactive material]</li>
	<li>Quickly air dry the membrane on a filter paper, seal it in a plastic bag and expose the membrane to an X-ray film at -20 &#176;C. Sufficient exposure time ranges between 5-30 days.</li>
</ol>
5. Confirmation of PRMT Expression and Equal Loading of Substrate
<ol>
	<li>After achieving sufficient exposures, transfer the membrane to a plastic box containing blocking buffer [Tris-buffered saline with Tween-20 (TBST + 3% non-fat milk)] and gently rock for 1 hr.</li>
	<li>Discard the blocking buffer as radioactive waste. Incubate the membrane with anti-HA antibody (1:1,000 dilution) in TBST overnight at 4 &#176;C, with gentle rocking.</li>
	<li>Wash the membrane with TBST 3x&#160;and incubate the membrane with HRP-labeled anti-mouse secondary antibody (1:5,000 dilution) in TBST at room temperature for 1 hr with gentle rocking.</li>
	<li>After 1 hr, wash the membrane with TBST 3x, and perform chemiluminiscence detection of proteins with an HRP substrate.</li>
	<li>After detection of the HA-tagged PRMTs, incubate the membrane with Ponseau-S staining solution for 5 min at room temperature. Quickly wash the membrane 3x with water to detect the GST-tagged substrates. Scan the image.</li>
</ol>

<ol>
	<li>Paik, W. K., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+methylase+I.+Purification+and+properties+of+the+enzyme.">Protein methylase I. Purification and properties of the enzyme.</a> J Biol Chem. 243, 2108-2114 (1968).</li><li>Lin, W. J., Gary, J. D., Yang, M. C., Clarke, S., Herschman, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+immediate-early+TIS21+protein+and+the+leukemia-associated+BTG1+protein+interact+with+a+protein-arginine+N-methyltransferase.">The mammalian immediate-early TIS21 protein and the leukemia-associated BTG1 protein interact with a protein-arginine N-methyltransferase.</a> J Biol Chem. 271, 15034-15044 (1996).</li><li>Boffa, L. C., Karn, J., Vidali, G., Allfrey, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+NG,+NG,-dimethylarginine+in+nuclear+protein+fractions.">Distribution of NG, NG,-dimethylarginine in nuclear protein fractions.</a> Biochem Biophys Res Commun. 74, 969-976 (1977).</li><li>Bedford, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+methylation+at+a+glance.">Arginine methylation at a glance.</a> J Cell Sci. 120, 4243-4246 (2007).</li><li>Lee, Y. H., Stallcup, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+protein+arginine+methylation+of+nonhistone+proteins+in+transcriptional+regulation.">Minireview: protein arginine methylation of nonhistone proteins in transcriptional regulation.</a> Mol Endocrinol. 23, 425-433 (2009).</li><li>Bedford, M. T., Clarke, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+arginine+methylation+in+mammals:+who,+what,+and+why.">Protein arginine methylation in mammals: who, what, and why.</a> Mol Cell. 33, 1-13 (2009).</li><li>Bikkavilli, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dishevelled3+is+a+novel+arginine+methyl+transferase+substrate.">Dishevelled3 is a novel arginine methyl transferase substrate.</a> Scientific reports. 2, 805 (2012).</li><li>Bikkavilli, R. K., Malbon, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+methylation+of+G3BP1+in+response+to+Wnt3a+regulates+{beta}-catenin+mRNA.">Arginine methylation of G3BP1 in response to Wnt3a regulates {beta}-catenin mRNA.</a> J Cell Sci. 124, 2310-2320 (2011).</li><li>Bikkavilli, R. K., Malbon, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt3a-stimulated+LRP6+phosphorylation+is+dependent+upon+arginine+methylation+of+G3BP2.">Wnt3a-stimulated LRP6 phosphorylation is dependent upon arginine methylation of G3BP2.</a> J Cell Sci. , (2012).</li><li>Tini, M., Naeem, H., Torchia, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+analysis+of+arginine+methylation+in+transcription.">Biochemical analysis of arginine methylation in transcription.</a> Methods Mol Biol. 523, 235-247 (2009).</li><li>Lee, J., Cheng, D., Bedford, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+in+protein+methylation.">Techniques in protein methylation.</a> Methods Mol Biol. 284, 195-208 (2004).</li><li>Cheng, D., Vemulapalli, V., Bedford, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+applied+to+the+study+of+protein+arginine+methylation.">Methods applied to the study of protein arginine methylation.</a> Methods Enzymol. 512, 71-92 (2012).</li><li>Zurita-Lopez, C. I., Sandberg, T., Kelly, R., Clarke, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+protein+arginine+methyltransferase+7+(PRMT7)+is+a+type+III+enzyme+forming+omega-NG-monomethylated+arginine+residues.">Human protein arginine methyltransferase 7 (PRMT7) is a type III enzyme forming omega-NG-monomethylated arginine residues.</a> J Biol Chem. 287, 7859-7870 (2012).</li></ol>

The authors have nothing to disclose.

The protocol described herein is routinely used to establish the methylation status of the identified PRMT substrates. This robust, and consistent assay will also provide definitive evidence on the specificity of PRMTs for their substrates. The key components for the success of this assay are: 1. Expression of PRMTs in mammalian cells, 2. Activity of purified PRMTs, 3. Expression and purification of substrate, and 4. Complete western transfer of the proteins to the nitrocellulose membrane.
Rec...

Protein methylation was first described in 19681. It was not until the first cloning of PRMT1 in 1996, that researchers began to appreciate the importance of this post-translational modification2. Interestingly, about 2% of arginine residues in the proteins of nuclear extracts are methylated3, indicating the abundance of this modification. Arginine is a positively charged amino acid with a basic side chain and the nitrogen/s within the side chains of arginine can be post-translationally modified via the addition of a methyl group, a process known as arginine methylation4-6. Arginine methylation is catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs). Arginines can either be monomethylated or dimethylated and the latter can be either symmetric or asymmetric depending on the type of PRMTs catalyzing the process4-6.
Proteins that display arginine-glycine rich motifs are potential targets for PRMT-mediated catalysis. PRMT-mediated methylation of substrates has been shown to modulate protein-protein interaction, protein-nucleic acid interaction, protein function, gene expression, and/or cellular signaling, all of which are critical for normal cellular homeostasis7-9. In order to understand the biological role of protein arginine methylation, precise, efficient, and reproducible assays are required to establish the methylation status of the identified PRMT substrates.
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 methylation10-12. 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 cells10,11. This in vitro methylation assay, as detailed in the protocol section, is based upon a method originally described by Tini and colleagues10. In this protocol, we show in detail the steps involved in the expression and purification of PRMT1 in mammalian cells. The ability of the purified PRMT1 to catalyze methylation on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate8, was later evaluated in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. Using this assay, we can reliably define the abilities of the PRMTs to methylate novel or known substrates, which is a primary step in the study of protein arginine methylation.

<table><tbody><tr><td>S-adenosyl-L-[methyl-&lt;sup&gt;3&lt;/sup&gt;H] methionine</td><td>Perkin Elmer</td><td></td><td></td></tr><tr><td>Ecoscint ultra&amp;nbsp;</td><td>National Diagnostics</td><td>LS-270</td><td></td></tr><tr><td>Bottle top dispenser</td><td>National Diagnostics</td><td>LS-900</td><td></td></tr><tr><td>AutoFluor</td><td>National Diagnostics</td><td>LS-315</td><td></td></tr><tr><td>6 ml Scintillation Vials</td><td>National Diagnostics</td><td>SKU:SVC-06</td><td></td></tr><tr><td>GST-sepharose</td><td>GE Life Sciences</td><td></td><td></td></tr><tr><td>L-Glutathione Reduced</td><td>Sigma</td><td>G4251</td><td></td></tr><tr><td>Lipofectamine</td><td>Invitrogen</td><td></td><td>Transfection reagent</td></tr><tr><td>Beas2B</td><td>ATCC</td><td></td><td></td></tr><tr><td>Optimem</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>NP-40</td><td>US-Biologicals</td><td></td><td></td></tr><tr><td>SDS</td><td>Sigma</td><td></td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma</td><td></td><td></td></tr><tr><td>PMSF</td><td>Sigma</td><td></td><td></td></tr><tr><td>anti-HA affinity matrix</td><td>Roche</td><td></td><td></td></tr><tr><td>Tris</td><td>Sigma</td><td></td><td></td></tr><tr><td>Nacl</td><td>Sigma</td><td></td><td></td></tr><tr><td>Bio-rad gel doc</td><td>Bio Rad</td><td></td><td></td></tr><tr><td>anti-HA antibody</td><td>roche</td><td></td><td></td></tr><tr><td>Ponseau S</td><td>Fisher Scientific</td><td></td><td></td></tr></tbody></table>

in vitro methylation assay to study protein arginine methylation

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-3H] methionine). Here we describe a step-by-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 S-adenosyl-L-[methyl-3H] 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.

The abilities of HA-PRMT1 to methylate GST-G3BP1 were determined by an in vitro methylation assay. HA-tagged PRMT1 purified from Beas2B cell lysates was employed in a methylation reaction containing GST-G3BP1, and 1 &#956;Ci of S-adenosyl-L-[methyl-3H] methionine, as a methyl donor. PRMT1 could efficiently catalyze the methylation of GST-G3BP1 (Figure 1, upper panel). Methylation reactions using pull-downs performed on lysates of empty vector transfected Beas2B cells served as a negat...

Watch this Scientific Journal Video about In vitro Methylation Assay to Study Protein Arginine Methylation at JoVE.com

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

in-vitro-methylation-assay-to-study-protein-arginine-methylation

Research

JoVE Journal

Biology

15.2K Views.  University of Illinois at Chicago.  Protein arginine methylatie, gekatalyseerd door een klasse van enzymen nl. Proteïne arginine methyl transferase (PRMTs), het proces van enzymatische additie van methylgroep (en) arginines in eiwitten. De in-vitro-methylatie test is de meest betrouwbare instrument voor de beoordeling van de methylatie status van bekende of nieuwe PRMT substraten. 

Video: In vitro Methylation Assay to Study Protein Arginine Methylation

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and ...

Genetics

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

Cancer Research

Proximity Ligation Assay Allows the Detection, Localization, and Quantification of Protein Arginine Methylation in Fixed Tissue

Arginine methylation is emerging as a key post-translational modification involved in a large range of biological processes. Its study in tissue is often limited by the lack of a specific antibody recognizing the target arginine residue. Proximity ligation assay (PLA) was originally developed to study protein/protein interactions. Here, we describe in detail a PLA protocol dedicated to the detection of arginine methylation that we applied to the glucocorticoid receptor (GR). Having previously shown that PRMT5 dimethylates GRs in cells, we used PLA with a pan symmetrical dimethyl antibody and an anti-GR antibody to measure GR methylation in breast tumors. We demonstrate that PLA offers a unique approach to measure arginine methylation of a target protein, even when the site of methylation has not been identified. This technique could be extended to other post-translational modifications where effective pan antibodies are available. Hence, we detail the PLA technology used to detect arginine methylation in fixed tissue using GR as an example.

Proximity ligation assay is a very useful technique to localize and quantify arginine methylation of a given protein when the modified arginine residue is unknown and/or if no specific antibody is available.

Arginine methylation is emerging as a key post-translational modification involved in a large range of biological processes. Its study in tissue is often ...

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

Biochemistry

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells

Protein methyltransferases (PRMTs) catalyze the transfer of a methyl group to arginine residues of substrate proteins. The PRMT family consists of nine members that can monomethylate or symmetrically/asymmetrically dimethylate arginine residues. Several antibodies recognizing different types of arginine methylation of various proteins are available; thus, providing tools for the development of PRMT activity biomarker assays. PRMT antibody-based assays are challenging due to overlapping substrates and motif-based antibody specificities. These issues and the experimental setup to investigate the arginine methylation contributed by individual PRMTs are discussed. Through the careful selection of the representative substrates that are biomarkers for eight out of nine PRMTs, a panel of PRMT activity assays were designed. Here, the protocols for cellular assays quantitatively measuring the enzymatic activity of individual members of the PRMT family in cells are reported. The advantage of the described methods is their straightforward performance in any lab with cell culture and fluorescent western blot capabilities. The substrate specificity and chosen antibody reliability were fully validated with knockdown and overexpression approaches. In addition to detailed guidelines of the assay biomarkers and antibodies, information on the use of an inhibitor tool compound collection for PRMTs is also provided.

These protocols provide the methodology used to assess the enzymatic activity of individual members of the protein arginine methyltransferase (PRMT) family in cells. Detailed guidelines on assessing PRMT activity using endogenous and exogenous biomarkers, methyl-arginine recognizing antibodies, and inhibitor tool compounds are described.

Protein methyltransferases (PRMTs) catalyze the transfer of a methyl group to arginine residues of substrate proteins. The PRMT family consists of nine ...

In vitro Methylation Assay to Study Protein Arginine Methylation

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