This work was supported by the French National Research Agency (ANR-14-CE02-0010). We thank Dr Benjamin Field and Dr. Elodie Lanet for critical reading of the manuscript. We thank Mr. Michel Terese for his help with video editing.

1. Preparation of 20 to 50% (w/v) Sucrose Gradients
Note: The gradients are made of 4 layers of sucrose (50%, 35% and 2 layers of 20%) in a 13.2 ml ultracentrifuge tube. In our experience, pouring the 20% sucrose in two separate layers greatly improves the quality of polysome preparations.
<ol>
	<li>Prepare the stock solutions. Ensure that all solutions are RNAse and DNAse free.
	<ol>
		<li>Prepare 10X Salt Solution: 400 mM Tris-HCl pH 8.4, 200 mM KCl and 100 mM MgCl2.</li>
		<li>Prepare 2 M Sucrose Solution: For 200 ml, dissolve 137 g of sucrose in 1X Salt Solution.</li>
	</ol>
	</li>
	<li>Dilute the Sucrose Solution in Salt Solution, as described in Table 1, to prepare the gradients. The volumes given are for six gradients.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Final sucrose concentration</td>
			<td>Sucrose 2M (ml)</td>
			<td>Salt solution 1X (ml)</td>
			<td>Final Vol. (ml)</td>
		</tr>
		<tr>
			<td>50%</td>
			<td>8.8</td>
			<td>3.2</td>
			<td>12</td>
		</tr>
		<tr>
			<td>35%</td>
			<td>12.9</td>
			<td>12.1</td>
			<td>25</td>
		</tr>
		<tr>
			<td>20%</td>
			<td>7.4</td>
			<td>17.6</td>
			<td>25</td>
		</tr>
		<tr>
			<td>20%</td>
			<td>5.8</td>
			<td>14.2</td>
			<td>20</td>
		</tr>
	</tbody>
</table>
Table 1. Dilutions of the Sucrose Solution to prepare six gradients.
<ol start="3">
	<li>Pour the layers as per Table 2.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sucrose layer</td>
			<td>50%</td>
			<td>35%</td>
			<td>20%</td>
			<td>20%</td>
		</tr>
		<tr>
			<td>Vol. (ml)</td>
			<td>1.85</td>
			<td>3.65</td>
			<td>3.65</td>
			<td>1.35</td>
		</tr>
	</tbody>
</table>
Table 2. Volume of sucrose solution per layer.
<ol start="4">
	<li>After pouring each layer, keep the tubes in a -40 &#176;C or -80 &#176;C freezer until complete freezing before adding the next sucrose layer. Freezing is usually achieved after 2 hr at -40 &#176;C, but waiting about 6 hr before pouring the next layer is recommended. The last 20% layer can be frozen or added freshly on the day of the experiment. 
	Note: Freezing each layer before adding the next one prevents from any mixing or disturbance of the layers. Gradients can be kept in a -40 &#176;C or -80 &#176;C freezer for at least six months.</li>
	<li>If it has not already been done, add the last 20% layer to the gradients on the day of the experiment. Then, let the gradients thaw out in a cold room or a fridge.</li>
</ol>
2. Preparation of Cytosolic Extracts
Note: We recommend using two gradients per biological sample. 300 mg is the optimum amount of plant material to prepare two gradients when working with 6 days old Arabidopsis thaliana seedlings. When working with less translationally active tissues, the amount of plant material can be increased up to 600 mg.
<ol>
	<li>Harvest six day old seedlings grown on &#189; Murashige and Skoog8 medium supplemented with 1% sucrose or equivalent media by quick-freezing in liquid nitrogen</li>
	<li>Grind plant material in a precooled mortar and pestle with liquid nitrogen.</li>
	<li>Weigh 300 mg of powdered material in a precooled weighing dish. Perform this step quickly to avoid thawing of the sample. 
	Note: Keep the frozen samples for no more than one week in a -80 &#176;C freezer.</li>
	<li>Add 2.4 ml of precooled polysome buffer (Salt Solution 4X, 5.26 mM EGTA, 0.5% (v/v) Octylphenoxy poly(ethyleneoxy)ethanol, branched, 50 &#181;g.ml-1 cycloheximide, 50 &#181;g.ml1 chloramphenicol) and homogenize by mixing with the pipette tip. Transfer into two 1.5 ml tubes. Work quickly to prevent the samples from warming.</li>
	<li>Centrifuge at 16,000 x g for 15 min at 4 &#176;C in a microcentrifuge to pellet debris. 
	Note: To prevent RNA degradation, always keep the samples at 4&#176;C, use RNase/DNase free solutions and work in RNase/DNase free conditions. Heparin can be added to the polysome buffer, to a final concentration of 300 &#181;g.ml-1, to enhance RNA protection. However, since heparin can interfere with downstream analysis, it will have to be removed during the RNA precipitation step by performing lithium chloride precipitation instead of ethanol precipitation (cf. note 4.7).</li>
</ol>
3. Polysome Profiling
<ol>
	<li>Carefully pipette the supernatant without disturbing the pellet. If plant fragments have been pipetted, repeat step 2.5. Load the supernatant on top of the gradient by gently pipetting onto the sidewall of the tube in a constant stream. Use one gradient for each 1.5 ml tube.</li>
	<li>Transfer to precooled buckets and centrifuge at 175,000 x g for 2 hr 45 min at 4 &#176;C, in an ultracentrifuge (e.g. 32,000 rpm when using a SW41 rotor).</li>
	<li>Set up the gradient collection system as described in Figure 1. The UV cuvette has a 1 mm pathlength.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/54231/54231fig1.jpg" /> 
Figure 1.&#160;Gradient collection system.&#160;The UV cuvette is connected by polyvinyl chloride tubing to a glass capillary tube that descends to the bottom of the gradient. The gradient progresses through the system thanks to a peristaltic pump. OD260 is continuously read and 2 ml fractions are collected. <a href="https://www.jove.com/files/ftp_upload/54231/54231fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Adjust the fraction collector to RT, and set the carousel at a speed that will allow the collection of 2 ml fractions. Use pre-cooled collection tubes.</li>
	<li>Collect the fractions from bottom to top using a glass capillary tube connected by polyvinyl chloride tubing to the gradient collection system. Use a plastic paraffin film to secure the connection between the polyvinyl chloride tubing and the glass capillary tube.</li>
	<li>Read the absorbance at 260 nm from the bottom to the top of the gradient. When the entire gradient is collected, place the collection tubes on ice before RNA extraction.</li>
</ol>
4. RNA Extraction
<ol>
	<li>Pool the fractions collected from two gradients, in 50 ml capped centrifuge tubes, as follows: 
	Polysomes: fractions 1 to 3 (12 ml) 
	Monosomes: fraction 4 (4 ml) 
	Supernatant: fractions 5 and 6 (8 ml)</li>
	<li>To each fraction, add 1 vol. 8M guanidine hydrochloride, 50 &#181;g acryl carrier and 1.5 vol. isopropanol. 
	Note: Acryl carrier is linear acrylamide, used as a coprecipitant to improve the recovery of nucleic acids during alcohol precipitation.</li>
	<li>Mix by inverting the tubes and precipitate O/N&#160;at -20 &#176;C.</li>
	<li>Centrifuge at 175,000 x g for 1 hr at 4 &#176;C, in an ultracentrifuge (32,000 rpm in a SW32 rotor). If the precipitation step is performed in a tube which is not compatible with ultracentrifugation, transfer to a suitable tube.</li>
	<li>Discard supernatant and dissolve the pellet in 200 &#181;l RNAse-free TE buffer (10 mM Tris-HCl pH 7.5 - 1 mM EDTA). Transfer to a fresh 1.5 ml tube.</li>
	<li>Extract RNA by adding 1 vol. water saturated phenol (pH 6.6) and 1 vol. chloroform:isoamyl alcohol (24:1). Mix vigorously. Centrifuge at 15,000 x g for 20 min at 4 &#176;C. Transfer the aqueous phase to a new 1.5 ml tube. 
	Note: Acid phenol (pH 4.5) can be used in order to minimize DNA contamination.</li>
	<li>Precipitate RNA by adding 1/10 vol. 3 M sodium acetate and 3 vol. 100% ethanol. Allow precipitation at -80 &#176;C for 20 min or O/N at -20 &#176;C. Centrifuge at 15,000 x g for 15 min at 4 &#176;C. 
	Note: If using heparin in the polysome buffer (cf. note 2.5), perform a lithium chloride (LiCl) precipitation instead of ethanol precipitation to properly remove any trace of heparin that could interfere with downstream analysis9. After extraction, add LiCl to a final concentration of 2.5 M. Allow precipitation for 30 min at -20 &#176;C or O/N at 4 &#176;C. Centrifuge at 15,000 x g for 15 min at 4 &#176;C.</li>
	<li>Wash the pellet with 500 &#181;l 75% ethanol. Air dry the pellet and dissolve in 30 &#181;l TE buffer. Assess RNA quality by electrophoresis on an RNAse free 1.2% agarose gel (100V, 15 min) or by capillary electrophoresis methods.</li>
</ol>
5. Data Analysis
<ol>
	<li>Export raw data to a graphic and data analysis software. 
	Note: As shown in Figure 2A and B, the raw profiles give qualitative information: the number of polysome peaks that can be seen, the height of the monosome peak, and the presence of a shoulder corresponding to the free ribosome subunits.</li>
	<li>Merge profiles to allow comparison of the profiles between samples. Use the screen reader tool to determine the X value of the monosome peak for each sample and align the monosome peaks by removing extra data points at the beginning of the curves. Identify the lowest point of the curve and determine its Y value (using the screen reader tool). Set the lowest point Y value to 0 by using the &#34;set columns value&#34; function.</li>
	<li>Determine the polysomes/monosomes ratio by integrating the area under the curve from raw profiles (Figure 3C). Use the data selector tool to define the border of the area to be integrated. Then use the integrate function (Analysis-Mathematics).</li>
	<li>Normalize the curves to the monosome peak by dividing all data points of a curve by the Y value of the summit of the monosome peak (determined using the screen reader tool). Select the column, then under Analysis-Mathematics, select Normalize and choose the &#34;divided by a specified value&#34; methods. This step allows comparison of the relative level of polysomes (Figure 3D).</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/54231/54231fig2.jpg" /> 
Figure 2. Representative polysome profiles. Arabidopsis thaliana wild-type (wt, ecotype Col-0) and mutant seedlings were grown for six days on &#189; Murashige and Skoog medium under long day photoperiods (16 hr light, 8 hr dark). A: raw polysome profiles from wt seedlings. B: raw polysome profiles from mutant seedlings. C: Determination of the percentage of polysomes and monosomes by integration of the area under the curve D:Polysome profiles normalized to the monosome peak. <a href="https://www.jove.com/files/ftp_upload/54231/54231fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<ol><li>Nelson, C. J., Millar, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+turnover+in+plant+biology%5BTitle%5D)+AND+%22Nat+Plants%22%5BJournal%5D)">Protein turnover in plant biology.</a> Nat Plants. 1 (3), 15017(2015).</li>
<li>Warner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+economics+of+ribosome+biosynthesis+in+yeast%5BTitle%5D)+AND+%22Trends+Biochem+Sci%22%5BJournal%5D)">The economics of ribosome biosynthesis in yeast.</a> Trends Biochem Sci. 24 (11), 437-440 (1999).</li>
<li>Amthor, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+McCree-de+Wit-Penning+de+Vries-Thornley+Respiration+Paradigms%3A+30+Years+Later%5BTitle%5D)+AND+%22Ann+Bot%22%5BJournal%5D)">The McCree-de Wit-Penning de Vries-Thornley Respiration Paradigms: 30 Years Later.</a> Ann Bot. 86 (1), 1-20 (2000).</li>
<li>Preiss, T., W Hentze, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Starting+the+protein+synthesis+machine%3A+eukaryotic+translation+initiation%5BTitle%5D)+AND+%22BioEssays+news+and+reviews+in+molecular%2C+cellular+and+developmental+biology%22%5BJournal%5D)">Starting the protein synthesis machine: eukaryotic translation initiation.</a> BioEssays news and reviews in molecular, cellular and developmental biology. 25 (12), 1201-1211 (2003).</li>
<li>Vogel, C., Marcotte, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insights+into+the+regulation+of+protein+abundance+from+proteomic+and+transcriptomic+analyses%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">Insights into the regulation of protein abundance from proteomic and transcriptomic analyses.</a> Nat Rev Genet. 13 (4), 227-232 (2013).</li>
<li>Baerenfaller, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-scale+proteomics+reveals+Arabidopsis+thaliana+gene+models+and+proteome+dynamics%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Genome-scale proteomics reveals Arabidopsis thaliana gene models and proteome dynamics.</a> Science. 320 (5878), 938-941 (2008).</li>
<li>Zanetti, E., Chang, I., Gong, F., Galbraith, D. W., Bailey-Serres, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunopurification+of+Polyribosomal+Complexes+of+Arabidopsis+for+Global+Analysis+of+Gene+Expression%5BTitle%5D)+AND+%22Plant+physiol%22%5BJournal%5D)">Immunopurification of Polyribosomal Complexes of Arabidopsis for Global Analysis of Gene Expression.</a> Plant physiol. 138 (2), 624-635 (2005).</li>
<li>Murashige, T., Skoog, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+revised+medium+for+rapid+growth+and+bioassays+with+tobacco+tissue+cultures%5BTitle%5D)+AND+%22Physiol+plant%22%5BJournal%5D)">A revised medium for rapid growth and bioassays with tobacco tissue cultures.</a> Physiol plant. 15 (3), 473-497 (1962).</li>
<li>del Prete, M. J., Vernal, R., Dolznig, H., Müllner, E. W., Garcia-Sanz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+polysome-bound+mRNA+from+solid+tissues+amenable+for+RT-PCR+and+profiling+experiments%5BTitle%5D)+AND+%22RNA%22%5BJournal%5D)">Isolation of polysome-bound mRNA from solid tissues amenable for RT-PCR and profiling experiments.</a> RNA. 13 (3), 414-421 (2007).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Arabidopsis+protocols%22">Arabidopsis protocols</a>. Salinas, J., Sanchez-Serrano, J. J. , Methods in molecular biology; 323. Clifton, N.J. (2006).</li>
<li>Piques, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ribosome+and+transcript+copy+numbers%2C+polysome+occupancy+and+enzyme+dynamics+in+Arabidopsis%5BTitle%5D)+AND+%22Mol+syst+biol%22%5BJournal%5D)">Ribosome and transcript copy numbers, polysome occupancy and enzyme dynamics in Arabidopsis.</a> Mol syst biol. 5 (314), 314(2009).</li>
<li>Morita, M., Alain, T., Topisirovic, I., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polysome+Profiling+Analysis%5BTitle%5D)+AND+%22bio-protocol%22%5BJournal%5D)">Polysome Profiling Analysis.</a> bio-protocol. 3 (14), 3-8 (2013).</li>
<li>Yángüez, E., Castro-Sanz, A. B., Fernández-Bautista, N., Oliveros, J. C., Castellano, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+genome-wide+changes+in+the+translatome+of+Arabidopsis+seedlings+subjected+to+heat+stress%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Analysis of genome-wide changes in the translatome of Arabidopsis seedlings subjected to heat stress.</a> PloS One. 8 (8), (2013).</li>
<li>Sormani, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sublethal+cadmium+intoxication+in+Arabidopsis+thaliana+impacts+translation+at+multiple+levels%5BTitle%5D)+AND+%22Cell+Physiol%22%5BJournal%5D)">Sublethal cadmium intoxication in Arabidopsis thaliana impacts translation at multiple levels.</a> Cell Physiol. 52 (2), 436-447 (2011).</li>
<li>Lanet, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biochemical+evidence+for+translational+repression+by+Arabidopsis+microRNAs%5BTitle%5D)+AND+%22Plant+cell%22%5BJournal%5D)">Biochemical evidence for translational repression by Arabidopsis microRNAs.</a> Plant cell. 21 (6), 1762-1768 (2009).</li>
<li>Jabnoune, M., Secco, D., Lecampion, C., Robaglia, C., Shu, Q., Poirier, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Rice+cis-Natural+Antisense+RNA+Acts+as+a+Translational+Enhancer+for+Its+Cognate+mRNA+and+Contributes+to+Phosphate+Homeostasis+and+Plant+Fitness%5BTitle%5D)+AND+%22Plant+cell%22%5BJournal%5D)">A Rice cis-Natural Antisense RNA Acts as a Translational Enhancer for Its Cognate mRNA and Contributes to Phosphate Homeostasis and Plant Fitness.</a> Plant cell. 25 (10), 4166-4182 (2013).</li>
<li>Ingolia, N. T. Genome-wide translational profiling by ribosome footprinting. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aIngolia%2C+NT+%22Methods+Enzymol%22">Methods Enzymol</a>. 470 (10), Elsevier Inc. (2010).</li>
<li>Juntawong, P., Girke, T., Bazin, J., Bailey-Serres, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Translational+dynamics+revealed+by+genome-wide+profiling+of+ribosome+footprints+in+Arabidopsis%5BTitle%5D)+AND+%22PNAS%22%5BJournal%5D)">Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis.</a> PNAS. 111 (1), 203-212 (2013).</li>
<li>Ingolia, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ribosome+profiling%3A+new+views+of+translation%2C+from+single+codons+to+genome+scale%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">Ribosome profiling: new views of translation, from single codons to genome scale.</a> Nat Rev Genet. 15 (3), 205-213 (2014).</li>
</ol>

The authors have nothing to disclose

The protocol we present here is an easy and cheap method for generating polysome profiles and isolating mRNAs associated with polysomes, single ribosomes or free of ribosomes. A wide range of different polysome fractionation methods is described in the literature. The method we have described here has been optimized to keep only the necessary compounds and has been adapted for plant material. In particular, we reduced the amount of detergent11 and added chloramphenicol to the buffer to fix the chloroplastic ri...

Protein synthesis is an essential and energetically costly process in all cells 1. First of all, cells must invest energy in the production of the translation machinery, the ribosomes. For example an actively dividing yeast cell produces as much as 2,000 ribosomes per minute. Such a production requires up to 60% of the total transcriptional activity and up to 90% of the total splicing activity of the cell 2. In addition, energy is required for the synthesis of amino acids, aminoacyl-tRNA and peptide bonds. In plants, adding one amino acid to a peptide chain costs from 4.5 to 5.9 molecules of ATP 3. Therefore, it is not surprising that the translation of mRNA to protein is a major site of regulation, particularly when it comes to dealing with changing environmental conditions.
The initiation step of translation, that is the association of a mRNA with the ribosome, is the main target of the regulation of translation4. As a consequence of the regulation of translation as well as other post-transcriptional regulatory steps, only 40% of the variations in protein concentration can be explained by mRNA abundance 5,6. Thus, the study of total mRNA gives relatively poor information about protein abundance. On the other hand, the association of mRNA with ribosomes gives better insight into protein abundance by giving access to those mRNAs involved in translation. Actively translated mRNAs are associated with several ribosomes in structures called polysomes. Conversely, poorly translated mRNAs will be associated with only one ribosome (monosome). Consequently, the translational status of an mRNA can be evaluated by monitoring its association with ribosomes7.
This protocol describes the isolation of polysomes from six days old Arabidopsis thaliana seedlings, the subsequent isolation of RNA, and the analysis of the results. Polysomes and monosomes are separated through a sucrose density gradient. Gradients are collected into six fractions. Some of the fractions are pooled to obtain three well separated fractions: polysomes, monosomes and the light fraction (hereafter called supernatant), which contains the free 60S and 40S ribosomal subunits and mRNAs that are not associated with ribosomes. Global translation activity can be estimated by generating a polysome/monosome ratio, which is determined by integration of the area under the curve, and by comparing the polysomes profiles. mRNAs and proteins are then extracted from the different fractions and used for analysis by RT-PCR, qRT-PCR, Northern blot, microarray, western blot or proteomics. This protocol has been validated for other plants and tissues.
The equipment required to perform this protocol are commonly found in most laboratories: There is no need for a gradient maker. Freezing each layer before adding the next one prevents from any mix or disturbance of the layers. No tube piercer is used for gradient collection which can be achieved by immersion of a glass capillary tube in the gradient. Therefore, the costly ultracentrifuge tubes remain undamaged and can be re-used many times. Collectively, this makes the present protocol an easy and cheap method for polysome profiling.

<table><tbody><tr><td>Ultracentrifuge tube, thinwall, polyallomer - 13.2 ml</td><td>Beckman Coulter</td><td>331372</td><td></td></tr><tr><td>Ultracentrifuge tube, thinwall, polyallomer - 38.5 ml</td><td>Beckman Coulter</td><td>326823</td><td></td></tr><tr><td>Glass capillary tube</td><td>Drummond Scientific</td><td>1-000-1000</td><td></td></tr><tr><td>Ultracentrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td>Optima series</td><td></td></tr><tr><td>Ultracentrifuge Rotor SW41</td><td>Beckman Coulter</td><td>331362</td><td></td></tr><tr><td>Ultracentrifuge Rotor SW32</td><td>Beckman Coulter</td><td>369650</td><td></td></tr><tr><td>Peristaltic pump</td><td>Any</td><td></td><td></td></tr><tr><td>Tygon R3607 polyvinyl chloride tubing&amp;nbsp;</td><td>Fisher Scientific</td><td>070534-22</td><td>Polyvinyl chloride tubing, 2.29 mm&amp;nbsp;</td></tr><tr><td>Fraction collector Model 2110</td><td>Bio-Rad</td><td>731-8120</td><td></td></tr><tr><td>UV cuvette</td><td>Hellma</td><td>170.700-QS</td><td>Quartz flow-through cuvette</td></tr><tr><td>UV Spectrophotometer</td><td>Varian</td><td>Cary50</td><td>Read every 0.0125 sec</td></tr><tr><td>All chemicals</td><td>Any</td><td></td><td>Use only Molecular Biology Grade</td></tr><tr><td>Murashige and Skoog Basal Salt Mixture (MS)</td><td>Sigma-Aldrich</td><td>M5524</td><td></td></tr><tr><td>Rnase-Free water</td><td>Any</td><td></td><td></td></tr><tr><td>Petri Dishes</td><td>Fisher Scientific</td><td>10083251&amp;nbsp;</td><td></td></tr><tr><td>Octylphenoxy poly(ethyleneoxy)ethanol, branched (Nonidet P40)</td><td>Euromedex</td><td>UN3500</td><td></td></tr><tr><td>Linear acrylamide (acryl carrier)</td><td>ThermoFischer scientific</td><td>AM9520</td><td>RNA precipitation carrier</td></tr><tr><td>OriginPro 8</td><td>OriginLab</td><td></td><td>Analysis software</td></tr></tbody></table>

an easy method for plant polysome profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

In the literature, polysome profiles are often shown from the light fraction to the heavy fraction as a result of the way the gradients are collected, i.e. from the top to the bottom. Since in the protocol described here the gradients are collected from the bottom to the top, the profiles we show start with the heavy fraction (the polysomes) and go to the light fraction (free ribosome subunits and RNAs) (Figure 2A). We then collect each gradient in six 2 ml fractions, but...

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An Easy Method for Plant Polysome Profiling

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

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12.6K Views.  Aix-Marseille Université. This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Video: An Easy Method for Plant Polysome Profiling

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Biochemistry

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

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.

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 ...

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells.  A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results.  This procedure is readily adaptable to mammalian cells.

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

Polysome Purification from Soybean Symbiotic Nodules

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper describes methods optimized to isolate plant-derived polyribosomes and their associated mRNAs to be analyzed using RNA-sequencing. First, cytoplasmic lysates are obtained through homogenization in polysome- and RNA-preserving conditions from whole, frozen soybean nodules. Then, lysates are cleared by low-speed centrifugation, and 15% of the supernatant is used for total RNA (TOTAL) isolation. The remaining cleared lysate is used to isolate polysomes by ultracentrifugation through a two-layer sucrose cushion (12% and 33.5%). Polysome-associated mRNA (PAR) is purified from polysomal pellets after resuspension. Both TOTAL and PAR are evaluated by highly sensitive capillary electrophoresis to meet the quality standards of sequencing libraries for RNA-seq. As an example of a downstream application, after sequencing, standard pipelines for gene expression analysis can be used to obtain differentially expressed genes at the transcriptome and translatome levels. In summary, this method, in combination with RNA-seq, allows the study of the translational regulation of eukaryotic mRNAs in a complex tissue such as the symbiotic nodule.

This protocol describes a method for eukaryotic polysome purification from intact soybean nodules. After sequencing, standard pipelines for gene expression analysis can be used to identify differentially expressed genes at the transcriptome and translatome levels.

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper ...

Developmental Biology

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell types. This balance is tightly controlled by gene expression that is fine-tuned at multiple levels, including transcription, post-transcription and translation. In this regard, a growing body of evidence highlights a critical role of translational regulation in coordinating neural stem cell fate decisions. Polysome fractionation is a powerful tool for the assessment of mRNA translational status at both global and individual gene levels. Here, we present an in-house polysome profiling pipeline to assess translational efficiency in cells from the developing mouse cerebral cortex. We describe the protocols for sucrose gradient preparation, tissue lysis, ultracentrifugation and fractionation-based analysis of mRNA translational status.

The development of the mammalian brain requires proper control of gene expression at the level of translation. Here, we describe a polysome profiling system with an easy-to-assemble sucrose gradient-making and fractionation platform to assess the translational status of mRNAs in the developing brain.

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell ...

Neuroscience

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

An Easy Method for Plant Polysome Profiling

Summary

Explore More Videos

mRNA Interactome Capture from Plant Protoplasts

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Polysome Profiling without Gradient Makers or Fractionation Systems

Eukaryotic Polyribosome Profile Analysis

Polysome Purification from Soybean Symbiotic Nodules

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling