Name: an easy method for plant polysome profiling
Uploaded: 2016-08-28T14:00:00
Description: Watch this Scientific Journal Video about An Easy Method for Plant Polysome Profiling at JoVE.com

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>...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="735">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge tube, thinwall, polyallomer - 13.2 ml</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">331372</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge tube, thinwall, polyallomer - 38.5 ml</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">326823</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Glass capillary tube</td>
			<td style="width:109px;">Drummond Scientific</td>
			<td style="width:132px;">1-000-1000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge&#160;</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">Optima series</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge Rotor SW41</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">331362</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge Rotor SW32</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">369650</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Peristaltic pump</td>
			<td style="width:109px;">Any</td>
			<td style="width:132px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Tygon R3607 polyvinyl chloride tubing&#160;</td>
			<td>Fisher Scientific</td>
			<td>070534-22</td>
			<td>Polyvinyl chloride tubing, 2.29 mm&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Fraction collector Model 2110</td>
			<td style="width:109px;">Bio-Rad</td>
			<td style="width:132px;">731-8120</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">UV cuvette</td>
			<td style="width:109px;">Hellma</td>
			<td style="width:132px;">170.700-QS</td>
			<td>Quartz flow-through cuvette</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">UV Spectrophotometer</td>
			<td style="width:109px;">Varian</td>
			<td style="width:132px;">Cary50</td>
			<td>Read every 0.0125 sec</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">All chemicals</td>
			<td style="width:109px;">Any</td>
			<td style="width:132px;"></td>
			<td>Use only Molecular Biology Grade</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Murashige and Skoog Basal Salt Mixture (MS)</td>
			<td>Sigma-Aldrich</td>
			<td>M5524</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Rnase-Free water</td>
			<td style="width:109px;">Any</td>
			<td style="width:132px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>10083251&#160;</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:233px;">Octylphenoxy poly(ethyleneoxy)ethanol, branched (Nonidet P40)</td>
			<td style="width:109px;">Euromedex</td>
			<td style="width:132px;">UN3500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Linear acrylamide (acryl carrier)</td>
			<td style="width:109px;">ThermoFischer scientific</td>
			<td style="width:132px;">AM9520</td>
			<td>RNA precipitation carrier</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">OriginPro 8</td>
			<td style="width:109px;">OriginLab</td>
			<td style="width:132px;"></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...

Watch this Scientific Journal Video about An Easy Method for Plant Polysome Profiling at JoVE.com

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 ...

Hi, I'm Cecile. I work at the LGBP in Marseilles. The protocol we are going to describe in this video is a simple method to isolate polysomes and monosomes from various plant tissues in order to identify mRNAs that are actively translated and to study translation regulation.As an example, we are going to show you the isolation of polysomes from six days'old Arabidopsis thaliana seedlings, subsequent isolation of RNA and the analysis of the results. Sow the seeds on plate and leave them two days at four degrees. Then grow the plants during six days.Harvest the seedling and grind them in liquid nitrogen. Weight 300 milligrams of plant powder and add 2.4 milliliter of polysome buffer. Centrifuge to collect plant fragments and load the supernatant on the top of a sucrose gradient.Ultra-centrifuge gradient then collect it from the bottom to the top with continuous reading of the OD.Pull the fraction into a polysome, a monosome and a supernatant fraction and precipitate RNA overnight. Ultra-centrifuge, resuspend the pellet and extract RNA for subsequent analysis. A few days before performing polysome profiling, prepare the sucrose gradient.Prepare 50, 35 and 20%sucrose solution and keep them at four degrees. Pour the 50%sucrose layer. Freeze it in a minus 40 freezer.After six hours, add the 35%layer and freeze it in a minus 40 freezer overnight. Pour the first 20%layer and freeze it. On the day of the experiment, remove the necessary number of gradients out of the freezer.Add the last 20%sucrose layer and let the gradients thaw out in a cold room. Prepare the sample we freshly collected plant. Here, we are using six days'old seedlings of Arabidopsis thaliana.Harvest the liquid nitrogen frozen plants and grind them thoroughly. Weight 300 milligrams of plant powder. Quickly add 2.4 milliliter of fresh polysome buffer and homogenize.Pipette the solution into two 1.5 milliliter tubes. Centrifuge the cytosolic extract. Pipette the supernatant.Be careful to avoid plant fragment. They would spoil the profile in the next step. Load the supernatant on the sucrose gradient without disrupting the gradient.Put the gradient in an SW 41 rotor bucket.Centrifuge. To visualize the polysome profile and collect the fraction, we use a simple system made of a capillary tube that plunge into the gradient. It is linked to a UV cuvette.It's self-connected to a peristaltic pump. A UV spectrophotometer registers continuously the OD as the gradient progress through the cuvette. The fraction collector allows collection of two milliliter fractions.Clean the cuvette and place it in the spectrophotometer. Remove the gradient from the bucket without disturbing it. Chlorophylls have to be concentrated on the top of the gradient.Plunge the capillary tubes to the bottom of the gradient. Start the peristaltic pump. The gradient is collected from the bottom to the top and OD is continuously read.Two milliliter fractions are collected. Here is the shape of a classical profile. The RNA precipitation is performed by using guanidinium hydrochloride and isopropanol.First, pour one volume of guanidinium hydrochloride in a 50 milliliter tube and then add the fraction. Then add 1.5 volume of isopropanol and 50 microgram equal courier. Precipitate the RNA overnight at minus 20 degrees.Transfer the fractions into 40 milliliter ultra-centrifuge tubes and balance the tube with isopropanol.Centrifuge. Remove the supernatant and air dry the pellet. Resuspend the pellet in 200 microliters of warm TE buffer.Further extract the RNA by performing a classical phenol chloroform extraction. A representative profile of polysomes extract from Arabidopsis six days'old seedling is shown here. The main peak corresponds to the monosome, the mRNA that are associated with a single ribosome.The first part of the profile corresponds to the polysomal fractions namely the mRNA associated with many ribosomes. Each peak match the association of the new ribosome to the mRNA. The last part of the profile corresponds to the so called supernatant fraction which contains the free ribosomal subunits and RNA.Large amount of 60S subunit form a shoulder next to the monosome peak. To assess their integrity, the RNAs extracted from the fractions can be run on a classical Aragose gel before being used for further experiments. We have used this protocol to isolate polysomes from Arabidopsis thaliana seedlings, but also from young and old rosettes as well as from Nicotiana benthamiana, tomato and rice leaves so it should work with a wide range of plants and tissues.The purified RNAs are compatible with and micro array analysis as well as for the identification of small RNAs associated with polysomal fractions.

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