Name: antibody-free assay for rna methyltransferase activity analysis
Uploaded: 2019-07-09T15:00:00.000+00:00
Duration: 8 min 31 s
Description: Watch this Scientific Journal Video about Antibody-Free Assay for RNA Methyltransferase Activity Analysis at JoVE.com

The authors would like to thank Dr. Turja Kanti Debnath for his help with ChemDraw. Research in the Xhemal&#231;e lab is supported by the Department Of Defense -&#160;Congressionally Directed Medical Research Program -&#160;Breast Cancer Breakthrough Award (W81XWH-16-1-0352), NIH Grant R01 GM127802 and start-up funds from the Institute of Cellular and Molecular Biology and the College of Natural Sciences at the University of Texas at Austin, USA.

1. In vitro transcription and gel purification of the target RNA
<ol>
	<li>Clone the sequence of interest into plasmids containing T7 and/or SP6 promoters using established molecular cloning techniques12 or kits.</li>
	<li>Linearizing the DNA template for in vitro transcription
	<ol>
		<li>Amplify the sequence of interest by PCR of the plasmid with primers designed to include the T7 promoter region through the insert, +20-30 bp upstream and downstream as previously described13.</li>
		<li>Alternatively, digest 5 &#181;g of the plasmid using a restriction enzyme (RE) cut site available downstream the insert according to the instructions provided by the supplier of the RE. 
		NOTE: Remember to double-check that the insert is not cut by the selected RE. Selection of the RE used in this step can dramatically affect the yield of the transcript. Preferentially, linearize with a blunt or 5&#39;-overhanging end producing RE to avoid template switching of the T7 RNA polymerase on the opposite DNA strand14. Try different RE if initial yields are insufficient.</li>
	</ol>
	</li>
	<li>Purify the resulting DNA using the kit for DNA agarose gel extraction and clean-up. Elute the DNA with 30 &#181;L of water.</li>
	<li>Mix well by vortexing, pipet 1.5 &#181;L and measure DNA concentration with a microvolume spectrophotometer.</li>
	<li>Use 250 ng of DNA to check the quality and size of the purified DNA on a 1% agarose gel electrophoresis in 1x TBE buffer migrated for 1 h at 4 V/cm.</li>
	<li>Thaw the frozen reagents of the in vitro transcription kit. Place the T7 RNA Polymerase Mix on ice, and the other reagents on a nutator at room temperature. Immediately after complete thawing, quick spin the rNTP tubes for 5 s to collect solution at the bottom of the tubes and place on ice. Keep the 10x Reaction Buffer at room temperature to avoid precipitation.</li>
	<li>Pipet into a 1.5 mL tube the following components of the 20 &#181;L transcription reaction at room temperature in the indicated order: 6 &#181;L of water, 2 &#181;L (0.1 - 1 &#181;g) of linear DNA template, 2 &#181;L of 75 mM ATP solution, 2 &#181;L of 75 mM GTP solution, 2 &#181;L of 75 mM CTP solution, 2 &#181;L of 75 mM UTP solution, 2 &#181;L of 10x Reaction Buffer and 2 &#181;L of T7 RNA Polymerase Mix. 
	NOTE: For DNA template generated by PCR, use 100 -&#160;200 ng DNA; for DNA template generated by restriction enzyme digest of a plasmid, use ~1 &#181;g. Active recombinant His6-tagged T7 RNA Polymerase can be purified in E. coli15.</li>
	<li>Mix the tube thoroughly. Quick spin for 5 s to collect reaction solution at the bottom of the tube, then incubate at 37 &#176;C for 2-4 h. 
	NOTE: Every transcript will behave differently depending on the DNA template, its length, its sequence or structure. Optimize in vitro transcription conditions by testing different incubation times up to 6 h; different concentrations of DNA template, T7 RNA Polymerase, or NTP; or addition of supplementary MgCl2 to what is present in the T7 RNA Polymerase Mix.</li>
	<li>At the end of the incubation period, add 1 &#181;L of DNase I per 20 &#181;L reaction and incubate at 37 &#176;C for 15 min. 
	NOTE: The transcription reaction can be temporarily kept on ice until the polyacrylamide gel is ready.</li>
	<li>Proceed with purification by polyacrylamide gel. 
	NOTE: Since RNA is sensitive to pH and RNase contamination, use RNA dedicated equipment and RNase-free reagents.</li>
	<li>Determine the percentage of polyacrylamide for the gel depending on the size of the transcript of interest: 3.5% for 100-2000 nt (XC: 460 nt ; BB: 100 nt), 5% for 80-500 nt (XC: 260 nt ; BB: 65 nt), 8% for 60-400 nt (XC: 160 nt ; BB: 45 nt), 12% for 40-200 nt (XC: 70 nt ; BB: 20 nt), 15% for 25-150 nt (XC: 60 nt ; BB: 15 nt), and 20% for 6-100 nt (XC: 45 nt ; BB: 12 nt).</li>
	<li>Prepare the urea denaturing polyacrylamide gel by combining the following reagents in a 50 mL conical tube: 9.6 g of molecular grade urea, 2 mL of 10x TBE, x mL of 40% acrylamide:bis-acrylamide mix (29:1) and water up to the 20 mL mark on the tube, where x=20 mL/(40%/gel percentage %). 
	CAUTION: Polyacrylamide gels often contain un-polymerized acrylamide which is a toxic material that can produce a hazard when introduced to the environment. Dispose of polyacrylamide gels through the institution&#8217;s chemical waste program.</li>
	<li>Microwave for 15 s at 30% power. Place on the nutator for 10 min at room temperature or until urea has completely dissolved.</li>
	<li>While the gel mixture is rotating, retrieve a gel cassette (18-well, 1 mm thick; 13.3 x 8.7 cm (W x L)), remove the comb and position the cassette for gel casting.</li>
	<li>Quick spin the 50 mL tube for 5 s to collect gel solution at the bottom of the tube. Add 125 &#181;L of 10% ammonium persulphate solution (APS). Place on the nutator for ~ 1 min. Quick spin again for 5 s.</li>
	<li>Add 25 &#181;L of tetramethylethylenediamine (TEMED) and carefully mix by pipetting up and down 5 times with a 25 mL pipette avoiding bubbles.</li>
	<li>Pipette into the gel cast and carefully insert the gel comb avoiding bubbles.</li>
	<li>Tighten the seal by adding a large binder clip over the top of the cassette.</li>
	<li>Allow the gel to polymerize for 1 h.</li>
	<li>Once the gel has solidified, remove the binder clip. Insert the cassette into the electrophoresis box. Add 1x TBE buffer into the top and bottom reservoirs.</li>
	<li>Retrieve transcription reaction. Add water up to 100 &#181;L, then add 100 &#181;L of 2x Gel Loading Buffer. Prepare the ladder by mixing the recommended amount with water to 10 &#181;L and 10 &#181;L of 2x Gel Loading Buffer to a separate 1.5 mL tube.</li>
	<li>Incubate both the ladder and the in vitro transcription reaction in the thermomixer at 70 &#176;C for 15 min.</li>
	<li>While the samples are denaturing at 70 &#176;C, carefully remove the comb, clean the wells by pipetting up and down each well with a P1000 pipette, and immediately pre-run for 10 min at 100 V.</li>
	<li>Once the samples have completed their 15 min denaturation at 70 &#176;C, remove the tubes from the thermomixer and immediately place on ice.</li>
	<li>Clean each well of the gel again with the P1000 pipette. Load 20 &#181;L of the ladder on the first well to the left, 20 &#181;L of the RNA sample on 10 separate wells, and 20 &#181;L of 1x Gel Loading Buffer on the unutilized wells.</li>
	<li>Run the gel at 100 V for 60 -240 min, depending on the % polyacrylamide of the gel. 
	NOTE: The dyes in the Gel Loading Buffer will separate into two bands as they traverse the gel, a slow migrating Bromophenol Blue (BB) blue band, and a fast migrating Xylene Cyanol (XC) cyan band. The approximate migration of these bands in different % polyacrylamide gels is well known (see step 1.11) and can be used to estimate the migration of RNA in the gel.</li>
	<li>Once the gel has been stopped, carefully remove the gel from the cassette.</li>
	<li>Place the gel in a clean box containing a solution of 50 mL of 1x TBE buffer with 50 &#181;L of nucleic acid gel stain and incubate for 5 min on a rocker to stain the RNA.</li>
	<li>Take a before and after band excision picture of the gel on the gel imaging system, preferably using blue light instead of UV transillumination.</li>
	<li>Excise each band of interest using a nuclease-free disposable gel cutting tip. After each well, transfer the tip containing the gel slice to a 1.5 mL tube and briefly spin to collect the gel slice. Repeat until all the bands have been collected in the same 1.5 mL tube.</li>
	<li>Once all the gel slices have been collected, add 100 &#181;L of nuclease-free water or TE buffer to the 1.5 mL tube. Store at 4 &#176;C for ~48 h. This allows the RNA to exit the gel slices into the solution.</li>
	<li>After 48 h, pipet the water or TE buffer to a fresh 1.5 mL tube. Dispose of the remaining gel slices. Purify the RNA via clean-up kit as follows.
	<ol>
		<li>Equilibrate the spin column at room temperature for at least 30 min.</li>
		<li>To the 100 &#181;L of RNA solution in the new 1.5 mL tube from step 1.32, add 350 &#181;L of RLT buffer and mix well for 2 min on a nutator. Spin for 1 s at 200 x g to collect solution at the bottom of the tube.</li>
		<li>Add 675 &#181;L of 100% EtOH and mix well for 2 min on the nutator. Spin for 1 s at 200 x g and immediately proceed to the next step.</li>
		<li>Transfer 565 &#181;L of the mixture onto the spin column and spin for 1 min at 15,000 x g. Empty the collection tube by aspiration.</li>
		<li>Repeat previous step with the second half of the sample.</li>
		<li>Add 500 &#181;L of RPE buffer to the column and spin for 1 min at 15,000 x g. Empty the collection tube by aspiration.</li>
		<li>Add 750 &#181;L of 80% ethanol to the column and spin for 1 min at 15,000 x g. Empty the collection tube by aspiration.</li>
		<li>Place the column in a new 2 mL collection tube with the lid open and spin at 15,000 x g for 5 min.</li>
		<li>Transfer the column to a new 1.5 mL tube.</li>
		<li>Add 17 &#181;L of water on the center of the column and spin at 15,000 x g for 1 min to elute. Elute again using another 17 &#181;L of water. The total recovered volume should be 32 &#181;L.</li>
	</ol>
	</li>
	<li>Mix well by vortexing, pipet 1.5 &#181;L and measure the concentration of the RNA using a microvolume spectrophotometer.</li>
	<li>Check the quality of the RNA purification by urea denaturing polyacrylamide gel as in steps 1.11-1.29.</li>
</ol>
2. In vitro RNA methyltransferase assay
<ol>
	<li>Set up the 100 &#181;L RNA methyltransferase assay in a 1.5 mL tube on ice as follows: 23 &#181;L of water, 10 &#181;L of 10x TBS (500 mM Tris-HCl, pH 7.5; 1.5 M NaCl), 2 &#181;L of 0.05 M EDTA, 5 &#181;L of 100 mM DTT, 40 &#181;L of 50% glycerol, 4 &#181;L of 58 &#181;M 3H-SAM, 5 &#181;L of 20x Protease Inhibitor Cocktail, 1 &#181;L of RNaseOUT (optional), 5 &#181;L of RNA and 5 &#181;L of Methyltransferase. 
	CAUTION: Radioactive tritiated material is hazardous and should only be handled while wearing gloves, a lab coat and any other necessary PPE. All pipette tips and tubes in contact with radioactive material are considered solid radioactive waste. Dispose of all solid and liquid radioactive waste according to the lab&#8217;s approved radioactive waste protocol. 
	NOTE: Include control samples without the methyltransferase and without the RNA. The reagent concentrations may require optimization and/or inclusion of divalent cation salts, such as MgCl2, or unlabeled SAM. The optimal range of final RNA concentration is from 50 nM to 1 &#181;M, while the methyltransferase concentration is from 25 nM to 300 nM.</li>
	<li>Mix thoroughly by gently vortexing the tube. Spin 5 s at 200 x g to collect the solution at the bottom of the tube. Incubate the tube(s) at 37 &#176;C for 2 h.</li>
	<li>Clean up the reaction using column purification as in step 1.32. 
	CAUTION: Be very careful to properly dispose of any radioactive materials, particularly during the column washes (pipette out instead of aspirating waste from collection tubes).</li>
	<li>Perform liquid scintillation count
	<ol>
		<li>Set up the scintillation count rack with one vial per sample, one vial for background measurement and one vial for the swipe test. Fill the vials with 5 mL of scintillation counting solution.</li>
		<li>Add 10 &#181;L of each eluted radioactive RNA sample into 1 vial, and tighten the lid and mix gently.</li>
		<li>Prepare the swipe test vials. Thoroughly rub cotton swabs on all surfaces and equipment used during the protocol. Add swabs to the vials filled with 5 mL of scintillation solution and tighten the lid.</li>
		<li>Run the samples on the scintillation counter as follows. Open the counter hood, insert the rack into the machine and close the hood. Select Count Single Rack. Select Select User Program. Select or create a program that measures tritium (3H) for 60 s. Hit Count Rack. Repeat the scintillation count three times. 
		NOTE: The equipment will measure the scintillation count of each sample and output to both the screen and a printout. Protocol can be paused here if desired. Remaining RNA samples from step 2.3 should be frozen at -80 &#176;C for later use.</li>
	</ol>
	</li>
	<li>Proceed with the autoradiogram
	<ol>
		<li>Prepare and pre-run urea denaturing polyacrylamide gel as in steps 1.11-1.20.</li>
		<li>Pipette 20 &#181;L of radioactive RNA material into a new 1.5 mL tube containing 20 &#181;L of 2x Gel Loading Buffer. Mix well. Prepare the ladder as in step 1.21. Incubate the samples at 70 &#176;C for 15 min.</li>
		<li>Wash the wells of the gel once more immediately before sample loading. Load 20 &#181;L of the prepared ladder, 20 &#181;L of the samples, and 20 &#181;L of 1x Gel Loading Buffer on remaining lanes. Run the gel at 100 V for 60-180 min, depending on polyacrylamide percentage.</li>
		<li>Once the gel finishes running, remove the gel from the cassette and place it in a box containing 50 mL of 1x TBE buffer with 5 &#181;L&#160;of ultrasensitive nucleic acid gel stain.</li>
		<li>Incubate for 5 min on the rocker to stain the RNA.</li>
		<li>Carefully take the gel out of the box and place it on the UV transilluminator of the gel imaging system with the wells up and the ladder on the left.</li>
		<li>Focus the camera on the gel, turn on the UV light and then take an image of the gel by exposing from 50 ms to 1 s depending on signal intensity.</li>
		<li>Turn off UV exposure, and save image as Tiff file.</li>
		<li>Place the gel back into the box. Remove TBE. Fix the gel with 50 mL of fixing solution (50% methanol, 10% acetic acid, 40% ultra-pure water) for 30 min at room temperature on a rocker.</li>
		<li>Gently move the gel again to a fresh black box containing 25 mL of the autoradiography enhancing solution. In the absence of a black box, cover the box with aluminum foil to protect the solution from light.</li>
		<li>Incubate for 30 min at room temperature on the rocker.</li>
		<li>Gently lift the gel and place it face-down on a sheet of plastic wrap with the wells up and the ladder on the right-hand side. Place two sheets of chromatography paper on the back of the gel. Gently flip the entire stack.</li>
		<li>Pre-heat the gel dryer to 80 &#176;C. Move back the plastic cover on the gel dryer. Insert wrap, gel and chromatography paper stack under the plastic cover and move the plastic cover back down to create a seal.</li>
		<li>Dry for 1 h at 80 &#176;C in the gel dryer.</li>
		<li>Turn off the gel dryer and gently remove the stack. Remove the wrap and second chromatography paper. Tape remaining chromatography paper with the dried gel in an autoradiogram cassette.</li>
		<li>Add 1 sheet of autoradiography film in the dark room.</li>
		<li>Place the cassette at -80 &#176;C and develop the film after 1 h to 4 weeks depending on signal intensity, which can be judged based on the previously measured scintillation counts: 1-4 weeks for 250-1,000 cpm, 24 h to 1 week for 1,000-10,000 cpm and 1 h to 24 h for &#62;10,000 cpm.</li>
		<li>Once the film is developed, place the film on top of the cassette and carefully mark with a lab marker the 4 edges of the gel, each well, and the position of the XC and BB dyes.</li>
		<li>Scan the film at 300 or 600 pixels per inch resolution and save the image as Tiff file.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Here, a simple and robust method for in vitro verification of RNA methyltransferase activity towards specific transcripts is reported. The assay takes advantage of the fact that S-adenosyl methionine can be tritiated on the methyl group donor (Figure 1), allowing for the methylated RNA to be accurately detected without the use of antibodies. However, it is important to note that this assay cannot indicate which residue or chemical group is methylated by the enzyme. To identify or to verify t...

DNA, RNA and proteins are subject to modifications that tightly regulate gene expression1. Among these modifications, methylations occur on all three biopolymers. DNA and protein methylations have been very well studied during the last three decades. In contrast, interest in RNA methylation has been only recently reignited in light of the important roles that proteins that write, erase or bind RNA methylations play in development and disease2. In addition to better known functions in the abundant ribosomal and transfer RNAs, RNA methylation pathways regulate specific messenger RNA stability3,4, splicing5 and translation6,7, miRNA processing8,9 and transcriptional pausing and release10,11.
Here, a simple and robust method for in vitro verification of RNA methyltransferase activity in the setting of a molecular biology lab is reported (summarized in Figure 1). Many studies assess the activity of an RNA methyltransferase through dot-blot with an antibody against the RNA modification of interest. However, dot-blot does not verify the integrity of the RNA upon incubation with the RNA methyltransferase. This is important because even minor contaminations of recombinant proteins with nucleases can lead to partial RNA degradation and confounding results. Moreover, even highly specific RNA modification antibodies can recognize unmodified RNAs with specific sequences or structures. The in vitro RNA methyltransferase assay reported here takes advantage of the fact that the S-adenosyl methionine can be tritiated on the methyl group donor (Figure 1), allowing for the methylated RNA to be accurately detected without the use of antibodies. Instructions are provided for in vitro transcription and purification of a transcript of interest and testing of the methylation of said transcript by the enzyme of interest. This method is flexible and robust, and can be adjusted according to the needs of any given project. For example, in vitro transcribed and purified RNAs, chemically synthesized RNAs, but also cellular RNAs can be used. This assay provides quantitative information in the form of scintillation counts, as well as qualitative information by showing where exactly the methylated RNA runs on a gel. This can provide unique insight into the function of an RNA methyltransferase, particularly when using cellular RNAs as a substrate, as it provides a method to directly observe the size of the RNA or RNAs that are targeted for methylation.

<table><tbody><tr><td>10 bp DNA Ladder</td><td>Invitrogen</td><td>10821-015</td><td>10 bp DNA Ladder kit.</td></tr><tr><td>10% Ammonium Persulfate (APS)&amp;nbsp;</td><td>N/A</td><td>N/A</td><td>For urea denaturing polyacrylamide gel (For 10 mL, dissolve 1g in 8 mL of milliQ water; adjust volume to 10 mL with milliQ water; filter the solution using a 10 mL syringe equiped with a 0.45 &amp;micro;m filter).</td></tr><tr><td>10X TBE Buffer</td><td>N/A</td><td>N/A</td><td>For urea denaturing polyacrylamide gel (For 1L, add 108 g of Tris Base, 55 g of Boric Acid to a cylinder with a stir bar; add 800 mL of distilled water and let dissolve; add 40mL of 0.5 M Na2EDTA (pH 8.0); adjust volume to 1L with milliQ water; filter the solution using a 0.22&amp;micro;m filter).</td></tr><tr><td>10X TBS</td><td>N/A</td><td>N/A</td><td>For 1L, add 60.5 g of Tris Base, 87.6 g of NaCl to a cylinder with a stir bar; add 800 mL of distilled water and let dissolve; adjust pH to 7.5 with concentrated HCl; adjust volume to 1L with milliQ water; filter the solution using a 0.22 &amp;micro;m filter.&amp;nbsp;</td></tr><tr><td>Acrylamide: Bis-Acrylamide 29:1 (40% Solution/Electrophoresis), Fisher BioReagents</td><td>Fisher</td><td>BP1408-1</td><td>For urea denaturing polyacrylamide gel.</td></tr><tr><td>ADENOSYL-L-METHIONINE, S-[METHYL-3H]; (SAM[3H])</td><td>Perkin Elmer</td><td>NET155V250UC</td><td>For in vitro methylation of RNA; Concentration = 1.0 mCi/mL; Specific activity = 17.1 Ci/mmol; Molarity=&lt;br/&gt; (1.0 Ci/L)/(83.2 Ci/mmol) = 0.0584 mmol/L = 58.4 &amp;micro;M.&amp;nbsp;&amp;nbsp; Upon receipt of the frozen 3H-SAM tube, thaw it at 4&amp;deg;C, make 20 &amp;micro;L aliquots, and freeze them at -30&amp;deg;C. Never refreeze and reuse a partially used aliquot.</td></tr><tr><td>Amersham Hypercassette Autoradiography Cassettes</td><td>GE Healthcare</td><td>RPN11649</td><td>For autoradiogram gel exposure.</td></tr><tr><td>Amersham Hyperfilm MP</td><td>GE Healthcare</td><td>28906846</td><td>For autoradiogram gel exposure.</td></tr><tr><td>Beckman Scintillation Counter</td><td>Beckman</td><td>LS6500</td><td>For liquid scintillation count.</td></tr><tr><td>Biorad Mini Horizontal Electrophoresis System</td><td>Biorad</td><td>1704466</td><td>Mini Horizontal Electrophoresis System.</td></tr><tr><td>cOmplete Mini EDTA-free Protease Inhibitor Cocktail Tablets</td><td>Roche Applied Science</td><td>4693159001</td><td>For a 20X solution, dissolve 1 tablet in 0.525 mL of nuclease free water.</td></tr><tr><td>Criterion Cell</td><td>Biorad</td><td>345-9902</td><td>RNase free empty cassette for polyacrylamide gel.</td></tr><tr><td>Criterion empty Cassettes</td><td>Biorad</td><td>1656001</td><td>Vertical midi-format electrophoresis cell.</td></tr><tr><td>DeNovix DS-11 Microvolume Spectrophotometer</td><td>DeNovix</td><td>DS-11-S</td><td>Microvolume Spectrophotometer for measuring DNA and RNA concentration.</td></tr><tr><td>Ecoscint Original</td><td>National Diagnostics</td><td>LS-271</td><td>For liquid scintillation count.</td></tr><tr><td>Fisherbrand 7mL HDPE Scintillation Vials</td><td>Fisher</td><td>03-337-1</td><td>For liquid scintillation count.</td></tr><tr><td>Fluoro-Hance-Quick Acting Autoradiography Enhancer</td><td>RPI CORP</td><td>112600</td><td>For autoradiogram gel pretreatment.</td></tr><tr><td>Gel dryer</td><td>Biorad</td><td>1651745</td><td>For drying gel.</td></tr><tr><td>Gel Loading Buffer II</td><td>Ambion</td><td>AM8547</td><td>For loading RNA in denaturing polyacrylamide urea gel (composition: 95% Formamide, 18 mM EDTA, and 0.025% SDS, Xylene Cyanol, and Bromophenol Blue).</td></tr><tr><td>GeneCatcher disposable gel excision tips</td><td>Gel Company</td><td>NC9431993</td><td>For removing bands from agarose and polyacrylamide gels.</td></tr><tr><td>Megascript Kit</td><td>Ambion</td><td>AM1333</td><td>For in vitro transcription with T7 RNA polymerase.&amp;nbsp;</td></tr><tr><td>Perfectwestern Extralarge Container</td><td>Genhunter Corporation</td><td>NC9226382 (clear)/ NC9965364 (black)</td><td>Gel staining box.</td></tr><tr><td>pRZ</td><td>Addgene</td><td>#27663</td><td>Plasmid for producing in vitro transcripts with homogeneous ends</td></tr><tr><td>Qiagen RNeasy MinElute Cleanup</td><td>Qiagen</td><td>74204</td><td>For RNA clean-up, use modified protocol provided in the protocol.</td></tr><tr><td>QIAquick Gel Extraction Kit (50)</td><td>Qiagen</td><td>28704</td><td>Kit for gel extraction and clean up of dsDNA fragment used for in vitro transcription.</td></tr><tr><td>Saran Premium Plastic Wrap</td><td>Saran Wrap</td><td>Amazon</td><td>For drying gel.</td></tr><tr><td>SYBR Gold</td><td>Invitrogen</td><td>S11494</td><td>Ultra sensitive nucleic acid gel stain.</td></tr><tr><td>SYBR Safe</td><td>Invitrogen</td><td>S33102</td><td>Nucleic acid gel stain.</td></tr><tr><td>TE</td><td>Sigma</td><td>93283-100ML</td><td>10 mM Tris-HCl, 1 mM disodium EDTA, pH 8.0</td></tr><tr><td>TEMED</td><td>Fisher</td><td>110-18-9</td><td>For urea denaturing polyacrylamide gel.</td></tr><tr><td>Thermomixer with SMARTBLOCK 24X 1.5mL TUBES</td><td>eppendorf</td><td>5382000023/5361000038</td><td>For temperature controlled incubation of 1.5 mL tubes.</td></tr><tr><td>TOPO TA Cloning Kit</td><td>life technologies</td><td></td><td>Kits for fast cloning of Taq polymerase&amp;ndash;amplified PCR products into vectors containing T7 and/or SP6 promoters for in vitro RNA transcription.</td></tr><tr><td>TURBO DNase (2 U&amp;frasl;&amp;micro;L)</td><td>Ambion</td><td>AM2238</td><td>For DNA removal from in vitro transcription reactions.</td></tr><tr><td>Urea</td><td>Sigma</td><td>51456-500G</td><td>For urea denaturing polyacrylamide gel.</td></tr><tr><td>Whatman 3MM paper</td><td>GE Healthcare</td><td>3030-154</td><td>Chromatography paper for drying gel.</td></tr></tbody></table>

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.

In vitro transcription reaction 
Figure 2A represents a typical run from an in vitro transcription reaction with the T7 RNA polymerase of the 7SK snRNA, which is a relatively short (331 nt) and highly structured RNA. As shown on that raw image, there are multiple undesired bands, both shorter and longer than 7SK, probably resulting from random transcriptional initiation or termination events. Because of this, gel purification following the in vitro tran...

Watch this Scientific Journal Video about Antibody-Free Assay for RNA Methyltransferase Activity Analysis at JoVE.com

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

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 ...

antibody-free-assay-for-rna-methyltransferase-activity-analysis

Research

JoVE Journal

Genetics

7.2K Views.  University of Texas at Austin. Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.

Video: Antibody-Free Assay for RNA Methyltransferase Activity Analysis

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

Biochemistry

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. 1, 2 It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. 2 A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, 3 Dictyostelium discoideum 4,5 and Drosophila. 6 Our recent work has shown that Ehmeth methylates tRNAAsp, and this finding indicates that this enzyme has a dual DNA/tRNAAsp methyltransferase activity. 7 This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. 8 The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

This protocol describes the preparation of a synthetic tRNA substrate for the Entamoeba histolytica DNA/tRNA methyltransferase 2 (Dnmt2) homolog Ehmeth and the measure of its methyltransferase activity. This experimental approach can be used for investigating the activity of other Dnmt2 proteins.

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of ...

Immunology and Infection

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects (TEdeff) in Cancers

We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription elongation (TE)&#8212;we call such cancers as TEdeff. Notably, TEdeff cancers are characterized by spurious transcription and faulty mRNA processing in a large set of genes, such as interferon/JAK/STAT and TNF/NF-&#954;B pathways, leading to their suppression. The TEdeff subtype of tumors in renal cell carcinoma and metastatic melanoma patients significantly correlate with poor response and outcome in immunotherapy. Given the importance of investigating TEdeff cancers&#8212;as it portends a significant roadblock against immunotherapy&#8212;the goal of this protocol is to establish an in vitro TEdeff mouse model to study these widespread, non-genetic transcriptional abnormalities in cancers and gain new insights, novel uses for existing drugs, or find new strategies against such cancers. We detail the use of chronic flavopiridol mediated CDK9 inhibition to abrogate phosphorylation of serine 2 residue on the C-terminal repeat domain (CTD) of RNA polymerase II (RNA Pol II), suppressing the release of RNA Pol II into productive transcription elongation. Given that TEdeff cancers are not classified under any specific somatic mutation, a pharmacological model is advantageous, and best mimics the widespread transcriptional and epigenetic defects observed in them. The use of an optimized sublethal dose of flavopiridol is the only efficacious strategy in creating a generalizable model of non-genetic widespread disruption in transcription elongation and mRNA processing defects, closely mimicking the clinically observed TEdeff characteristics. Therefore, this model of TEdeff can be leveraged to dissect, cell-autonomous factors enabling them in resisting immune-mediated cell attack.

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects TEdeff in Cancers

The protocol details an in vitro murine carcinoma model of non-genetic defective transcription elongation. Here, chronic inhibition of CDK9 is used to repress productive elongation of RNA Pol II along pro-inflammatory response genes to mimic and study the clinically observed TEdeff phenomenon, present in about 20% of all cancer types.

We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription ...

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and sequencing (MeRIP-seq) is a method that aims to enrich for methylated RNA and ultimately identify modified transcripts. Briefly, sonicated RNA is incubated with an antibody for 5-methylated cytosines and precipitated with the assistance of protein G beads. The enriched fragments are then sequenced and the potential methylation sites are mapped based on the distribution of the reads and peak detection. MeRIP can be applied to any organism, as it does not require any prior sequence or modifying enzyme knowledge. In addition, besides fragmentation, RNA is not subjected to any other chemical or temperature treatment. However, MeRIP-seq does not provide single-nucleotide prediction of the methylation site as other methods do, although the methylated area can be narrowed down to a few nucleotides. The use of different modification-specific antibodies allows MeRIP to be adjusted for the different base modifications present on RNA, expanding the possible applications of this method.

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

The methylated RNA immunoprecipitation assay is an antibody-based method used to enrich for methylated RNA fragments. Coupled with deep sequencing, it leads to the identification of transcripts carrying the m5C modification.

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and ...

DNAzyme-dependent Analysis of rRNA 2&#8217;-O-Methylation

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in higher eukaryotes may promiscuously recognize other RNA species and 2&#8217;-O-methylate multiple targets. Here, we provide step-by-step guide for the fast and non-expensive analysis of the site-specific 2&#8217;-O-methylation using a well-established method employing short DNA oligonucleotides called DNAzymes. These DNA fragments contain catalytic sequences which cleave RNA at specific consensus positions, as well as variable homology arms directing DNAzyme to its RNA targets. DNAzyme activity is inhibited by 2-&#8217;O-methylation of the nucleotide adjacent to the cleavage site in the RNA. Thus, DNAzymes, limited only by the consensus of the cleaved sequence, are perfect tools for the quick analysis of snoRNA-mediated RNA 2&#8217;-O-methylation. We analyzed snoRNA snR13- and snR47-guided 2&#8217;-O-methylation of 25S ribosomal RNA in Saccharomyces cerevisiae to demonstrate the simplicity of the technique and to provide a detailed protocol for the DNAzyme-dependent assay.

Here we present a protocol for DNAzyme-dependent cleavage of RNA. This enables fast and site-dependent analysis of RNA 2&#8217;-O-methylation. This approach can be used for the preliminary or major assessment of snoRNA activity.

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in ...

Developmental Biology

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

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 ...

Biology

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as epitranscriptomics. Among others, N6-methyladenosine (m6A) and 5-methylcytosine (m5C) RNA modifications have been described on mRNA molecules and may have a role in modulating cellular processes. Epitranscriptomics is thus a new layer of regulation that must be considered in addition to transcriptomic analyses, as it can also be altered or modulated by exposure to any chemical or biological agent, including viral infections.
Here, we present a workflow that allows analysis of the joint cellular and viral epitranscriptomic landscape of the m6A and m5C marks simultaneously, in cells infected or not with the human immunodeficiency virus (HIV). Upon mRNA isolation and fragmentation from HIV- infected and non-infected cells, we used two different procedures: MeRIP-Seq, an RNA immunoprecipitation-based technique, to enrich for RNA fragments containing the m6A mark and BS-Seq, a bisulfite conversion-based technique, to identify the m5C mark at a single nucleotide resolution. Upon methylation-specific capture, RNA libraries are prepared for high-throughput sequencing. We also developed a dedicated bioinformatics pipeline to identify differentially methylated (DM) transcripts independently from their basal expression profile.
Overall, the methodology allows exploration of multiple epitranscriptomic marks simultaneously and provides an atlas of DM transcripts upon viral infection or any other cell perturbation. This approach offers new opportunities to identify novel players and novel mechanisms of cell response, such as cellular factors promoting or restricting viral replication.

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in viral infections is just starting to be explored and could highlight new viral-host interaction mechanisms. In this work, we provide a pipeline to investigate m6A and m5C RNA modifications in the context of viral infections.

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as ...

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

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A Method for Measuring RNA N⁶-methyladenosine Modifications in Cells and Tissues

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects (TE^deff) in Cancers

Methylated RNA Immunoprecipitation Assay to Study m⁵C Modification in Arabidopsis

DNAzyme-dependent Analysis of rRNA 2’-O-Methylation

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

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

Exploring m⁶A and m⁵C Epitranscriptomes upon Viral Infection: an Example with HIV