Published: August 28th, 2016
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 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.
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 ....
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.
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.......
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.......
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.....
|Ultracentrifuge tube, thinwall, polyallomer - 13.2 ml
|Ultracentrifuge tube, thinwall, polyallomer - 38.5 ml
|Glass capillary tube
|Ultracentrifuge Rotor SW41
|Ultracentrifuge Rotor SW32
|Tygon R3607 polyvinyl chloride tubing
|Polyvinyl chloride tubing, 2.29 mm
|Fraction collector Model 2110
|Quartz flow-through cuvette
|Read every 0.0125 sec
|Use only Molecular Biology Grade
|Murashige and Skoog Basal Salt Mixture (MS)
|Octylphenoxy poly(ethyleneoxy)ethanol, branched (Nonidet P40)
|Linear acrylamide (acryl carrier)
|RNA precipitation carrier
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