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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Y., Xiao, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Integrated+polysome+profiling+and+ribosome+profiling+method+to+investigate+in+vivo+translatome.">An Integrated polysome profiling and ribosome profiling method to investigate in vivo translatome.</a> Methods in Molecular Biology. 1712, 1-18 (2018).</li><li>Chasse, H., Boulben, S., Costache, V., Cormier, P., Morales, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+translation+using+polysome+profiling.">Analysis of translation using polysome profiling.</a> Nucleic Acids Research. 45 (3), 15 (2017).</li><li>Aboulhouda, S., Di Santo, R., Therizols, G., Weinberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate,+streamlined+analysis+of+mrna+translation+by+sucrose+gradient+fractionation.">Accurate, streamlined analysis of mrna translation by sucrose gradient fractionation.</a> Bio-protocol. 7 (19), 2573 (2017).</li><li>Kudla, M., Karginov, F. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mRNA+translation+by+polysome+profiling.">Measuring mRNA translation by polysome profiling.</a> Methods in Molecular Biology. 1421, 127-135 (2016).</li><li>Faye, M. D., Graber, T. E., Holcik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+selective+mRNA+translation+in+mammalian+cells+by+polysome+profiling.">Assessment of selective mRNA translation in mammalian cells by polysome profiling.</a> Journal of Visualized Experiments: JoVE. 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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. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+footprint+profiling+of+translation+throughout+the+genome.">Ribosome footprint profiling of translation throughout the genome.</a> Cell. 165 (1), 22-33 (2016).</li><li>Ripmaster, T. L., Vaughn, G. P., Woolford, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DRS1+to+DRS7,+novel+genes+required+for+ribosome+assembly+and+function+in+Saccharomyces+cerevisiae.">DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.</a> Molecular and Cellular Biology. 13 (12), 7901-7912 (1993).</li><li>Lo, Y. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+biogenesis+in+the+yeast+Saccharomyces+cerevisiae.">Ribosome biogenesis in the yeast Saccharomyces cerevisiae.</a> Genetics. 195 (3), 643-681 (2013).</li><li>Klinge, S., Woolford, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+assembly+coming+into+focus.">Ribosome assembly coming into focus.</a> Nature Reviews. Molecular Cell Biology. 20 (2), 116-131 (2018).</li><li>Bassler, J., Hurt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+ribosome+assembly.">Eukaryotic ribosome assembly.</a> Annual Review of Biochemistry. 88, 281-306 (2019).</li><li>Kressler, D., Hurt, E., Bassler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+puzzle+of+life:+Crafting+ribosomal+subunits.">A puzzle of life: Crafting ribosomal subunits.</a> Trends in Biochemical Sciences. 42 (8), 640-654 (2017).</li><li>Prattes, M., Lo, Y. H., Bergler, H., 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=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 ...

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