Name: polysome profiling without gradient makers or fractionation systems
Uploaded: 2021-06-01T14:00:00
Description: Watch this Scientific Journal Video about Polysome Profiling without Gradient Makers or Fractionation Systems at JoVE.com

The authors thank Dr. Percy Tumbale and Dr. Melissa Wells for their critical reading of this manuscript. This work was supported by the US National Institute of Health Intramural Research Program; US National Institute of Environmental Health Sciences (NIEHS; ZIA ES103247 to R.E.S).

1. Preparation of 7% - 47% sucrose gradients
NOTE: The linear range of the sucrose gradient can be modified to achieve better separation depending on the cell type used. This protocol is optimized for polysome profiles for S. cerevisiae.
<ol>
	<li>Prepare stock solutions of 7% and 47% sucrose in sucrose gradient buffer (20 mM Tris-HCl pH 7.4, 60 mM KCl, 10 mM MgCl2, and 1 mM DTT). Filter sterilize the sucrose stock solutions through a 0.22 &#956;m filter and store at 4 &#...

<ol>
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L., Gorospe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+fractionation+to+analyze+mRNA+distribution+profiles.">Polysome fractionation to analyze mRNA distribution profiles.</a> Bio-protocol. 7 (3), 2126 (2017).</li><li>Chikashige, Y., 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=Gcn2+eIF2alpha+kinase+mediates+combinatorial+translational+regulation+through+nucleotide+motifs+and+uORFs+in+target+mRNAs.">Gcn2 eIF2alpha kinase mediates combinatorial translational regulation through nucleotide motifs and uORFs in target mRNAs.</a> Nucleic Acids Research. 48 (16), 8977-8992 (2020).</li><li>Ingolia, N. 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H., 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=Cryo-EM+structure+of+the+essential+ribosome+assembly+AAA-ATPase+Rix7.">Cryo-EM structure of the essential ribosome assembly AAA-ATPase Rix7.</a> Nature Communications. 10 (1), 513 (2019).</li><li>Pillon, M. C., Sobhany, M., Borgnia, M. J., Williams, J. G., Stanley, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grc3+programs+the+essential+endoribonuclease+Las1+for+specific+RNA+cleavage.">Grc3 programs the essential endoribonuclease Las1 for specific RNA cleavage.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (28), 5530-5538 (2017).</li><li>Woolford, J. L., Baserga, S. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+the+nascent+ribosome:+AAA-ATPases+in+eukaryotic+ribosome+biogenesis.">Shaping the nascent ribosome: AAA-ATPases in eukaryotic ribosome biogenesis.</a> Biomolecules. 9 (11), 715 (2019).</li><li>Pillon, M. C., Sobhany, M., Stanley, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+molecular+crosstalk+within+the+essential+Grc3/Las1+pre-rRNA+processing+complex.">Characterization of the molecular crosstalk within the essential Grc3/Las1 pre-rRNA processing complex.</a> RNA. 24 (5), 721-738 (2018).</li><li>Hu, W., Coller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+analysis+for+determining+mRNA+and+ribosome+association+in+Saccharomyces+cerevisiae.">Polysome analysis for determining mRNA and ribosome association in Saccharomyces cerevisiae.</a> Methods in Enzymology. 530, 193-206 (2013).</li><li>He, S. L., Green, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+analysis+of+mammalian+cells.">Polysome analysis of mammalian cells.</a> Methods in Enzymolog. 530, 183-192 (2013).</li><li>Anand, M., Chakraburtty, K., Marton, M. J., Hinnebusch, A. G., Kinzy, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interactions+between+yeast+translation+eukaryotic+elongation+factor+(eEF)+1A+and+eEF3.">Functional interactions between yeast translation eukaryotic elongation factor (eEF) 1A and eEF3.</a> The Journal of Biological Chemistry. 278 (9), 6985-6991 (2003).</li><li>Serikawa, K. 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Here a method to create polysome profiles without the use of expensive automated fractionation systems has been described. The advantage of this method is that it makes polysome profiling accessible to labs that do not have automated fractionation systems. The major disadvantages of this protocol are tedious hand fractionation and reduced sensitivity compared to the dedicated density fractionation system.
This protocol entails careful preparation of sucrose gradients with resolution sufficient...

Ribosomes are mega-Dalton ribonucleoprotein complexes that perform the fundamental process of translating mRNA into proteins. Ribosomes are responsible for carrying out the synthesis of all proteins within a cell. Eukaryotic ribosomes comprise two subunits designated as the small ribosomal subunit (40S) and the large ribosomal subunit (60S) according to their sedimentation coefficients. The fully assembled ribosome is designated as the 80S monosome. Polysomes are groups of ribosomes engaged in translating a single mRNA molecule. Polysome fractionation by sucrose density gradient centrifugation is a powerful method used to create ribosome profiles, identify specific mRNAs associated with translating ribosomes and analyze polysome associated factors1,2,3,4,5,6,7,8,9,10,11,12,13. This technique is often used to separate polysomes from single ribosomes, ribosomal subunits, and messenger ribonucleoprotein particles. The profiles obtained from fractionation can provide valuable information regarding the translation activity of polysomes14 and the assembly status of the ribosomes15,16,17.
Ribosome assembly is a very complex process facilitated by a group of proteins known as ribosome assembly factors18,19,20,21. These factors perform a wide range of functions during ribosome biogenesis through interactions with many other proteins, including ATPases, endo- and exo-nucleases, GTPases, RNA helicases, and RNA binding proteins22. Polysome fractionation has been a powerful tool used to investigate the role of these factors in ribosome assembly. For example, this method has been utilized to demonstrate how mutations in the polynucleotide kinase Grc3, a pre-rRNA processing factor, can negatively affect the ribosome assembly process17,23. Polysome profiling has also highlighted and shown how the conserved motifs within the ATPase Rix7 are essential to ribosome production16.
The procedure for polysome fractionation begins with making soluble cell lysates from cells of interest. The lysate contains RNA, ribosomal subunits, and polysomes, as well as other soluble cellular components. A continuous, linear sucrose gradient is made within an ultracentrifuge tube. The soluble fraction of cell lysate is gently loaded onto the top of the sucrose gradient tube. The loaded gradient tube is then subjected to centrifugation, which separates the cellular components by size within the sucrose gradient by the force of gravity. The larger components travel further into the gradient than the smaller components. The top of the gradient houses the smaller, slower traveling cellular components, whereas the larger, faster traveling cellular components are found at the bottom. After centrifugation, the contents of the tube are collected as fractions. This method effectively separates ribosomal subunits, monosomes, and polysomes. The optical density of each fraction is then determined by measuring the spectral absorbance at a wavelength of 254 nm. Plotting absorbance vs. fraction number yields a polysome profile.
Linear sucrose density gradients can be generated utilizing a gradient maker. Following centrifugation, gradients are often fractionated, and absorbances measured using an automated density fractionation system3,7,13,24,25. While these systems work very well to produce polysome profiles, they are expensive and can be cost-prohibitive to some laboratories. Here a protocol to generate polysome profiles without the use of these instruments is presented. Instead, this protocol utilizes equipment typically available in most molecular biology laboratories.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Automatic Fractionator</td>
			<td>Brandel</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clariostar Multimode Plate Reader</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma Aldrich</td>
			<td>C7698</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Invitrogen</td>
			<td>15508-013</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Beads, acid washed</td>
			<td>Sigma Aldrich</td>
			<td>G8772</td>
			<td>425&#8211;600 &#956;m</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma Aldrich</td>
			<td>H4784</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride, 1 M</td>
			<td>KD Medical</td>
			<td>CAC-5290</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle, 22 G, Metal Hub</td>
			<td>Hamilton Company</td>
			<td>7748-08</td>
			<td>custom length 9 inches, point style 3</td>
		</tr>
		<tr>
			<td>Optima XL-100K Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Centrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td>331372</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Test Tube Peg Rack</td>
			<td>Fisher Scientific</td>
			<td>14-810-54A</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 4 Fluorometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q33228</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit RNA HS Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q32855</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse Inhibitor</td>
			<td>Applied Biosystems</td>
			<td>N8080119</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>SW41 Swinging Bucket Rotor Pkg</td>
			<td>Beckman Coulter</td>
			<td>331336</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 3 mL</td>
			<td>Coviden</td>
			<td>888151394</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris, 1 M,&#160; pH 7.4</td>
			<td>KD Medical</td>
			<td>RGF-3340</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Star Microplate, 96 wells</td>
			<td>Greiner Bio-One</td>
			<td>655801</td>
			<td></td>
		</tr>
	</tbody>
</table>

polysome profiling without gradient makers or fractionation systems

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

Three representative polysome profiles are shown in Figure 3. All profiles are from the same yeast strain. A typical polysome profile will have well-resolved peaks for the 40S, 60S, and 80S ribosomal subunits as well as polysomes. The crest of each ribosomal subunit and polysome peak will be apparent on each profile (Figure 3). A representative profile from an automated density fractionation system is shown in Figure 3A. The sucrose...

Watch this Scientific Journal Video about Polysome Profiling without Gradient Makers or Fractionation Systems at JoVE.com

Polysome Profiling without Gradient Makers or Fractionation Systems

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...

This protocol describes how to generate a polysome profile which reveals molecular details about the activity of ribosomes inside the cell. This technique allows users to obtain the valuable information provided by a polysome profile, who do not have access to automated gradient fractionation systems. Take your time while preparing the gradients, and store the gradients in a location where they will not be disrupted by a compressor or other mechanical operations as they settle into a linear gradient.To begin, prepare stock solutions of 7 and 47%sucrose in sucrose gradient buffer. Filter sterilize the sucrose stock solutions through a 0.22 micron filter. Prepare 14 milliliters of 17, 27, and 37%sucrose solutions by dispensing and mixing the 7 and 47%stock solutions.Place six polypropylene centrifuge tubes into a full view test tube rack. Ensure that there is enough space between the tubes so that the actions with one tube do not disturb the others. Attach a nine inch, 22 gauge needle with a blunt tip to a three milliliter syringe, and perform a test fill and dispense to ensure that the syringe can hold the sucrose solution without any dripping before setting up the gradients.Add two milliliters of the 7%sucrose to the bottom of each centrifuge tube. Then, add two milliliters of the 17%sucrose beneath the 7%solution, by positioning the needle tip within the immediate vicinity of the tube bottom. Carefully and slowly dispense the solution.Repeat the procedure with two milliliters each of the 27, 37 and 47%sucrose solution, ensuring that each layer is distinguishable from the other by a line marking the separation of densities. Store the gradients at four degree Celsius overnight to allow them to settle into a continuous, increasing percentage of sucrose. After the growth and harvesting of yeast, Saccharomyces cerevisiae cells, resuspend the cells in chilled 700 microliters of polysome extraction buffer.Add 100 units of RNAse Inhibitor. Then, add 400 microliters of pre-chilled glass beads with an approximate size of 425 to 600 microns. Transfer the mixture to a 1.5 milliliter centrifuge tube with the glass beads, and disrupt the yeast cells by vigorous agitation in a bead-beater for five minutes.After disrupting the cells, clarify the lysate by centrifugation. Determine the concentration of the RNA in the clarified lysate by measuring the absorbance at 260 nanometers, using a fluorescence-based RNA detection system. Ensure that RNA concentration is 0.5 to one microgram per microliter.If the RNA concentration is too low, reduce the volume of extraction buffer used to resuspend the cells. Carefully load the lysate onto the top of the gradients. Place the pipette tip against the inner wall at the top of the polypropylene tube.Angle the tube and slowly dispense the lysate onto the top of the gradient, dribbling against the wall. Gently place the tubes into the pre-chilled buckets of a swinging bucket rotor, and centrifuge the gradients. After centrifugation, carefully removed the centrifuge tubes from the swinging bucket rotor, and place them in a tube holder.Label the 96-well plates to store the fractions and pre-chill on ice. Collect 100 or 200 microliter fractions starting from the top of the gradient, by carefully inserting a pipette tip into the top of the gradient, and collecting the fractions until the entire gradient is aliquoted. Measure the absorbance of each fraction at 254 nanometers with a spectrophotometer against the 7 and 47%sucrose solutions as blanks.Create the polysome profile by plotting the fraction number versus absorbance. Three representative polysome profiles from yeast S.Cerevisiae, are shown. The crest of each ribosomal subunit and polysome peak is apparent on each profile.A representative profile from an automated density fractionation system is shown here. This profile was produced from a continuous absorbance profile as the sucrose gradient was displaced from the bottom up by a chase solution, through a detector flow cell and collected in fractions. The polysome profile generated by hand fractionating 200 and 100 microliter samples is shown here.The most important thing to remember with this procedure is to not disrupt the gradients. Take care not to introduce air bubbles or disrupt the gradient by dispensing solution too rapidly. Following this procedure, RNA can be extracted from the gradients for further analysis to determine mRNAs that are associating with active ribosomes

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