An Easy Method for Plant Polysome Profiling

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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.

1. Prepare the stock solutions. Ensure that all solutions are RNAse and DNAse free.
   1. Prepare 10X Salt Solution: 400 mM Tris-HCl pH 8.4, 200 mM KCl and 100 mM MgCl_2.
   2. Prepare 2 M Sucrose Solution: For 200 ml, dissolve 137 g of sucrose in 1X Salt Solution.
2. Dilute the Sucrose Solution in Salt S....

Results

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.......

Discussion

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.......

Materials

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