Name: Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays (Video) | JoVE Journal
Uploaded: 2010-06-18T17:00:00.000+00:00
Duration: 7 min 32 s
Description: Learn to measure tRNA charging levels in Saccharomyces cerevisiae using microarray technology and periodate oxidation. This method enables genome-wide analysis of aminoacylation dynamics under stress conditions.

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5 minutes

The yeast work in the Pan lab was funded by a grant from Ajinomoto, Inc. Japan and National Institutes of Health Training Grant T32GM007183-33. We thank Dr. Ronald Wek at the Indiana University School of Medicine for long-term collaboration on the yeast projects. We also thank Dr. Kimberly Dittmar for developing the tRNA charging microarray method.

Part 1: Isolation of total charged tRNA
<ol>
<li>Pellet cells in a tabletop centrifuge at 2000 RFC for 5 minutes. The equivalent of 1 OD600 of cells should produce at least 1&mu;g of total RNA and a minimum of 30 &mu;g is recommended for the procedure.</li>
<li>Resuspend cells in lysis buffer solution consisting of 0.3 M buffered Na+-acetate (pH 4.5) and 10 mM EDTA. Vortex with an equal volume of Na+-acetate-saturated phenol/chloroform (pH 4.5) three times each for 30 s. Be sure to always keep samples on ice between vortexing. Buffered acetate can be prepared from NaOAC and HOAc while acetate saturated phenol/chloroform can be prepared by mixing 1 part 5 M buffered acetate (pH 4.5) with 9 parts phenol/chloroform. 
 Note: total RNA isolation is carried out under mildly acidic conditions and on ice to avoid deacylation of charged tRNA.</li>
<li>Centrifuge at 18,600 RFC for 15 minutes at 4&deg;C. Remove the aqueous layer and perform another acetate-saturated phenol/chloroform extraction.</li>
<li>After the second extraction precipitate the RNA by adding 2.7x volumes of ethanol and centrifuging at 18,600 RFC for 30 minutes at 4&deg;C. </li>
<li>Resuspend RNA pellets in lysis buffer solution and then precipitate with ethanol a second time. </li>
<li>Finally, resuspend the RNA in a solution containing 10 mM buffered acetate (pH 4.5) and 1 mM EDTA for subsequent treatment. The sample can be stably stored at -80&deg;C for about two weeks. Longer storage is not recommended as some charged tRNA will start to deacylate.</li>
</ol>
Part 2: Preparation of tRNA Standards
<ol>
 <li>Heat E. coli tRNA standards at 10.5 &mu;M to 85&deg;C in 45 mM Tris-Cl pH 7.5 for 2 minutes. Take the tube out and leave at room temperature for 3 minutes.</li>
 <li>Add MgCl2 to a final concentration of 20 mM and incubate at 37&deg;C for 5 minutes.</li>
 <li>Add the synthetase reaction mix so the final reaction contains 5 &mu;M tRNA, 60 mM Tris-HCl (pH 7.5), 12.5 mM MgCl2, 10 mM KCl, 3 mM dithiothreitol, 1.5 mM ATP, 1 mM spermine, 1 mM of the respective amino acid, and 4.2 units/&mu;l E. coli aminoacyl-tRNA synthetase mix. Incubate at 37&deg;C for 15 minutes.</li>
 <li>Add an equal volume of 0.5 M buffered acetate (pH 4.5) and extract with acetate-saturated phenol/CHCl3 at pH 4.5.</li>
 <li>Precipitate the tRNA standards with 2.7x volumes of ethanol and centrifuging at 18,600 RFC for 30 minutes at 4&deg;C. </li>
 <li>Resuspend the standards in 50 mM buffered acetate (pH 4.5) with 1 mM EDTA and store at &#8722;80&deg;C for up to one month.</li>
</ol>
Part 3: Cy3/Cy5 labeling of tRNA
<ol>
 <li>For the charged tRNA sample incubate total RNA at a concentration of 0.1 &mu;g/&mu;l with 0.066 &mu;M each E. coli tRNA standards (i.e. 0.67 pmole each standard per &mu;g total RNA) and 100 mM buffered acetate (pH 4.5) in the presence of 50 mM NaIO4 for 30 minutes at room temperature. For the control total tRNA sample use 50 mM NaCl in place of NaIO4. Remember to dilute the RNA only with buffered solutions to preserve charging. </li>
 <li>To quench the reaction add glucose to 100 mM and incubate at room temperature for 5 minutes.</li>
 <li>In order to remove any remaining NaIO4 from the sample perform a buffer exchange using a G25 spin column. For best results equilibrate the column first by running 200 mM buffered acetate buffer (pH 4.5) through it prior to applying your sample.</li>
 <li>Precipitate the sample by adding buffered acetate (pH 4.5) to a final concentration of 133 mM and NaCl to a final concentration of 66 mM and 2.7x volumes of ethanol. Occasionally a second precipitation may be needed for the oxidized samples. This is necessary only if the pellet looks significantly different than the control pellet of the same sample. For example a significantly bigger or more diffuse pellet. If a second ethanol precipitation is required resuspend pellet in 50 mM acetate buffer pH 4.5 and 200 mM NaCl before the addition of ethanol. </li>
 <li>For deacylation the tRNA samples are resuspended in 50 mM Tris-HCl (pH 9) and incubated at 37 &deg;C for 30 min. The reaction is neutralized by the addition of an equal volume of 50 mM buffered acetate (pH 4.5) and 100 mM NaCl. Precipitate with 2.7x volumes of ethanol. After precipitation, RNA is resuspended in water at ~1 &mu;g/&mu;l. Both the control and oxidized samples should be run on agarose gels to check RNA quality.</li>
 <li>To attach fluorescent oligo tags onto the tRNA 0.1 &mu;g/&mu;l deacylated RNA is incubated in 1x ligase buffer, 15% DMSO, 4 &mu;M Cy3- or Cy5-containing oligonucleotides, 0.5 units/&mu;l T4 DNA ligase, and yeast exo-phosphatase (5,000 units/&mu;l) at 16&deg;C overnight (over 16 h). The exo-phosphatase is necessary only for samples obtained from yeast and can be omitted if this protocol is used for E. coli or human samples. </li>
 <li>After ligation samples are mixed with 4 volumes of 50 mM KOAc (pH 7), 200 mM KCl, and then extracted with an equal volume of phenol/chloroform. Following extraction of the aqueous phase, RNA preparations are precipitated with ethanol and resuspended in water to approximately 0.1 &mu;g/&mu;l.</li>
 <li>Ligation efficiency can be assessed by running 5-10% of the samples on 12% polyacrylamide gels containing 7M urea and visualized with a fluorescent gel scanner. This PAGE analysis is also useful to determine the amount of oxidized and control samples needed for microarray hybridization. A good ligation result should show ~10% or more of the Cy3/Cy5 containing oligonucleotide being ligated to the tRNA. For samples with good labeling efficiency, 0.1-0.5 &mu;g total RNA per sample is used for array hybridization.</li>
</ol>
Part 4: Hybridization and Analysis of the Microarray
<ol>
 <li>Prior to hybridization microarray slides are boiled in distilled water for 1-2 minutes to remove unbound oligonucleotides.</li>
 <li>Cy3/Cy5 labeled samples are combined with 140 &mu;l of hybridization buffer and salmon sperm DNA to a final concentration of 140 &mu;g/ml and poly(A) to a final concentration of 70 &mu;g/ml.</li>
 <li>A hybridization program, (table 1), is run on an automatic array hybridizer. For tRNA work, it is highly recommended that an automatic hybridization machine be used since manual hybridization produces high background.</li>
 <li>After the program is complete manually wash the slides with Wash 3 solution at least twice by gently shaking in a 50 ml conical tube for 3-5 minutes.</li>
 <li>Slides can be dried by centrifuging at a low speed, less than 200 RFC, for 5-10 minutes. It is important to keep the slides out of the light since exposure to light will bleach the fluorophores.</li>
 <li>After the slides are dried they should be scanned immediately for optimal results; if necessary they can be saved for several days in a dry dark place at room temperature. Slides can be scanned 2-3 times without noticeable degradation in image quality.</li>
</ol>
Part 5: Representative Results
When isolated properly the total RNA sample should produce a very clean spectrum with an OD260:OD280 ratio near 2. There should be no detectable protein or phenol contamination. When run on 1% agarose gels, a strong tRNA band should be observed with other possible nucleic acid bands varying between organisms. After oxidation a clean tRNA band should be observed. If a sample is noticeably weaker than other samples or smearing is observed, samples should not be used for microarrays. Microarrays should have very low background which is an order of magnitude lower than the weakest tRNA probe. High background is usually due to problems during the washing steps. This can usually be fixed by preparing fresh wash solutions and thoroughly washing all equipment and tubing to remove residual and precipitated SDS.
<table border="1">
<tbody>
 <tr>
 <td>Step</td>
 <td>Details</td>
 </tr>
 <tr>
 <td>O-ring conditioning</td>
 <td>75&deg;C, 2 min</td>
 </tr>
 <tr>
 <td>Introduce sample</td>
 <td>60&deg;C</td>
 </tr>
 <tr>
 <td>Denature Sample</td>
 <td>90&deg;C, 5 min</td>
 </tr>
 <tr>
 <td>Hybridization</td>
 <td>60&deg;C, 16h</td>
 </tr>
 <tr>
 <td>Wash 1</td>
 <td>50&deg;C, flow 10s hold 20s</td>
 </tr>
 <tr>
 <td>Wash 2</td>
 <td>42&deg;C, flow 10s hold 20s</td>
 </tr>
 <tr>
 <td>Wash 3</td>
 <td>42&deg;C, flow 10s hold 20s</td>
 </tr>
 </tbody>
</table>
Table 1: Hybridization protocol

<ol><li>Zaborske, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+analysis+of+tRNA+charging+and+activation+of+the+eIF2+kinase+Gcn2p%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Genome-wide analysis of tRNA charging and activation of the eIF2 kinase Gcn2p.</a> J Biol Chem. 284 (37), 25254-25267 (2009).</li>
<li>Wek, S. A., Zhu, S., Wek, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+histidyl-tRNA+synthetase-related+sequence+in+the+eIF-2+alpha+protein+kinase+GCN2+interacts+with+tRNA+and+is+required+for+activation+in+response+to+starvation+for+different+amino+acids%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">The histidyl-tRNA synthetase-related sequence in the eIF-2 alpha protein kinase GCN2 interacts with tRNA and is required for activation in response to starvation for different amino acids.</a> Mol Cell Biol. , 4497-4506 (1995).</li>
<li>Hinnebusch, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Translational+Regulation+of+GCN4+and+the+General+Amino+Acid+Control+of+Yeast%5BTitle%5D)+AND+%22Annual+Review+of+Microbiology%22%5BJournal%5D)">Translational Regulation of GCN4 and the General Amino Acid Control of Yeast.</a> Annual Review of Microbiology. 59 (1), 407-450 (2005).</li>
<li>Braeken, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+horizons+for+%28p%29ppGpp+in+bacterial+and+plant+physiology%5BTitle%5D)+AND+%22Trends+in+Microbiology%22%5BJournal%5D)">New horizons for (p)ppGpp in bacterial and plant physiology.</a> Trends in Microbiology. 14 (1), 45-54 (2006).</li>
<li>Dong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Uncharged+tRNA+Activates+GCN2+by+Displacing+the+Protein+Kinase+Moiety+from+a+Bipartite+tRNA-Binding+Domain%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">Uncharged tRNA Activates GCN2 by Displacing the Protein Kinase Moiety from a Bipartite tRNA-Binding Domain.</a> Molecular Cell. 6 (2), 269-279 (2000).</li>
<li>Cashel, M. The Stringent Response. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCashel%2C+M+%22Escherichia+coli+and+Salmonella%3A+cellular+and+molecular+biology%22">Escherichia coli and Salmonella: cellular and molecular biology</a>. Neidhardt, F. C. , ASM Press. Washington DC. 1458-1496 (1996).</li>
<li>Dittmar, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+charging+of+tRNA+isoacceptors+induced+by+amino-acid+starvation%5BTitle%5D)+AND+%22EMBO+Rep%22%5BJournal%5D)">Selective charging of tRNA isoacceptors induced by amino-acid starvation.</a> EMBO Rep. 6 (2), 151-157 (2005).</li>
<li>Zhou, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+levels+of+tRNA+abundance+and+alteration+of+tRNA+charging+by+bortezomib+in+multiple+myeloma%5BTitle%5D)+AND+%22Biochem+Biophys+Res+Commun%22%5BJournal%5D)">High levels of tRNA abundance and alteration of tRNA charging by bortezomib in multiple myeloma.</a> Biochem Biophys Res Commun. 385 (2), 160-164 (2009).</li>
</ol>

No conflicts of interest declared.

We present a method for simultaneously determining the relative charging levels of all tRNAs at the genomic scale. The protocol presented here has been optimized for S. cerevisiae, and it has also been successfully used to measure tRNA charging profiles in E. coli and human cell cultures. It can be modified to accommodate any organism with known genome sequences (allowing for annotation of all tRNA genes) as long as total charged tRNA can be obtained.

<table><tbody><tr><td>Microspin G-25 columns</td><td>GE Healthcare</td><td>25-5325-01</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; tRNA&lt;sup&gt;Phe&lt;/sup&gt;</td><td>Sigma-Aldrich</td><td>R3143</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; tRNA&lt;sup&gt;Tyr&lt;/sup&gt;</td><td>Sigma-Aldrich</td><td>R0258</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; tRNA&lt;sup&gt;Lys&lt;/sup&gt;</td><td>Sigma-Aldrich</td><td>R6018</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; aminoacyl-tRNA synthetases</td><td>Sigma-Aldrich</td><td>A3646</td><td></td></tr><tr><td>T4 DNA ligase</td><td>USB Corp., Affymetrix</td><td>70042X</td><td></td></tr><tr><td>PerfectHyb Plus</td><td>Sigma-Aldrich</td><td>H-7033</td><td></td></tr><tr><td>Hyb4 station</td><td>Digilab</td><td></td><td></td></tr><tr><td>GenePix 4000B scanner</td><td>Axon instruments</td><td></td><td></td></tr><tr><td>GenePix 6.0</td><td>Axon instruments</td><td></td><td></td></tr></tbody></table>

genome-wide analysis of aminoacylation (charging) levels of trna using microarrays

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

The overall goal of the following experiment is to determine relative changes in TRNA charging levels. This is achieved by isolating RNA at a low pH before and after treatment to isolate TRNA with intact charging levels. As a second step, the RNA is subjected to a per iodate oxidation treatment, which allows for determination of relative charging levels.Next ligate samples to a fluorescent oligonucleotide and hybridize on a microarray in order to measure changes in ligation levels due to A-T-R-N-A charging results are obtained. The CHO genomic level changes in TRNA charging based on TRNA microarray technology. Hi, I'm John Zai from the laboratory of DRT Pan in the department of Biochemistry and molecular biology at the University of Chicago.Today I'll be showing you a procedure for isolating and measuring charge TRNA at the genomic level. We use this to measure changes in TNA charging during stressor puns in our lab. So let's get started.To isolate total charge to TRNA, start by growing a minimum of the equivalent of 30 OD 600 of yeast cells, which should produce 30 micrograms of total RNA pellet the cells in a tabletop centrifuge. Resuspend cells in a lysis buffer solution. Add an equal volume of sodium acetate, saturated phenyl chloroform and vortex for 30 seconds, followed by icing for at least 30 seconds.Repeat vortexing and dicing two more times. Centrifuge the sample at 18, 600 RFC for 15 minutes at four degrees Celsius. Then remove the aqueous layer and perform another acetate saturated phenyl chloroform extraction after the second extraction.Precipitate the RNA by adding 2.7 x volumes of ethanol and centrifusion at 18, 600 RFC for 30 minutes at four degrees Celsius. Resus suspend RNA pellets in lysis buffer solution and then precipitate with ethanol a second time. Finally, re suspend the RNA in a solution containing 10 millimolar buffered acetate and one millimolar EDTA for subsequent treatment.The sample can be stably stored at minus 80 degrees Celsius for about two weeks longer. Storage is not recommended as some charged TNA will start to deci isolate prior to labeling with S3 or SCI five fluorescent oligonucleotide tags. The total TRNA is subjected to a pariah day oxidation treatment, which allows for determination of relative charging levels mixed the total RNA with 0.066 micromolar of each eco irna standard and 100 millimolar buffered acetate in the presence of 50 millimolar sodium par IID date for the control TRNA sample that will not be oxidized use 50 millimolar sodium chloride in place of the sodium par IID date.Incubate the reactions for 30 minutes at room temperature after 30 minutes, quench the reaction by adding glucose to 100 millimolar and incubating at room temperature for five minutes. Once the reaction is quenched, perform a buffer exchange using a G 25 spin column to remove any remaining sodium prorate from the samples. Precipitate the samples by adding buffered acetate to a final concentration of 133 millimolar sodium chloride to a final concentration of 66 millimolar and 2.7 x volumes of ethanol.Next deci, isolate the tRNA samples by Resus, suspending them in 50 millimolar tris HCL and incubating at 37 degrees Celsius for 30 minutes. After 30 minutes, neutralize the reaction by adding an equal volume of 50 millimolar buffered acetate and 100 millimolar sodium chloride precipitate with 2.7 x volumes of ethanol. Re suspend the RNA pellet in water at about one microgram per microliter.Once the control and oxidized samples have been checked for RNA quality by running on agarose gels, we can proceed with the fluorescent labeling to attach fluorescent oligo tags onto the TRNA. Combine 0.1 microgram per microliter of de RNA with four micromolar SI three or SI five containing oligonucleotides ligase buffer 15%DMSO 0.5 units per microliter of T four DNA ligase and yeast exo phosphatase incubate at 16 degrees Celsius for over 16 hours After ligation, mix the samples with four volumes of potassium acetate and potassium chloride, and then extract with an equal volume of phenyl chloroform following extraction of the aqueous phase precipitate the RNA preparations with ethanol. Finally, resus, suspend the RNA pellets in water to approximately 0.1 microgram per microliter prior to hybridization oil, microarray slides and distilled water for one to two minutes to remove unbound oligonucleotides.Next, combine the S3 or SCI five labeled samples with 140 microliters of hybridization buffer salmon sperm DNA to a final concentration of 140 micrograms per milliliter and poly A to a final concentration of seven D micrograms per milliliter run a hybridization program on an automatic array hybridizer. We highly recommend the use of an automatic hybridization machine for TRNA work since manual hybridization produces high background. When the hybridization program is complete, manually wash the slides with wash three solution by gently shaking in a 50 milliliter conical tube for three to five minutes.Wash the slides at least twice in tubes pre-wrapped with aluminum foil. Dry the slides by centrifuging at a low speed for five to 10 minutes for optimal results. The slides should be scanned immediately after they're dried.Slides can be scanned two to three times without noticeable degradation in image quality representative results of a properly isolated total RNA sample and its analysis are shown here. This sample has a very clean spectrum with an OD two 60 to OD two 80 ratio near two with no detectable protein or phenol contamination when run on 1%agro gels. Both oxidized and control samples should show a strong TNA band with other possible nucleic acid bands varying between organisms.If a sample is noticeably weaker than other samples, or if smearing is observed, samples should not be used for microarrays. Microarrays should have a very low background, which is an order of magnitude lower than the weakest TRNA probe. I've just shown you how to isolate and determine the relative charging levels of TRNA when doing this procedure.It's important to remember to keep your reagents free of nucleases, especially during the oxidation and dilation steps. Thanks for watching and good luck with your experiments.

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Learn to measure tRNA charging levels in Saccharomyces cerevisiae using microarray technology and periodate oxidation. This method enables genome-wide analysis of aminoacylation dynamics under stress conditions.

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

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