En nem metode til Plant Polysomprofiler profilering
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
Proteinsyntese er en væsentlig og energisk kostbar proces i alle celler 1. Først og fremmest skal celler investere energi i produktionen af translationsmaskineriet, ribosomerne. For eksempel en deler sig aktivt gærcelle producerer så meget som 2.000 ribosomer per minut. En sådan produktion kræver op til 60% af den samlede transkriptionel aktivitet og op til 90% af den totale splejsning aktivitet af celle 2. Desuden er nødvendige energi til syntese af aminosyrer, aminoacyl-tRNA og peptidbindinger. I planter, tilsætning én aminosyre til en peptidkæde omkostninger fra 4,5 til 5,9 molekyler ATP 3. Derfor er det ikke overraskende, at oversættelsen af mRNA til protein er et stort site af regulering, især når det kommer til at håndtere skiftende miljøforhold.
Indledningen trin i oversættelse, der er foreningen af en mRNA med ribosomet, er det vigtigste mål for reguleringen afoversættelse 4. Som følge af reguleringen af oversættelse samt andre post-transkriptionelle regulatoriske trin, kan kun 40% af de variationer i koncentrationen protein forklares med mRNA overflod 5,6. Således studiet af totalt mRNA giver forholdsvis dårlig information om protein overflod. På den anden side, sammenslutningen af mRNA med ribosomer giver bedre indblik i protein overflod ved at give adgang til disse mRNA er involveret i oversættelsen. Aktivt oversatte mRNA'er er associeret med flere ribosomer i strukturer kaldet polysomer. Omvendt vil dårligt oversat mRNA være forbundet med kun én ribosom (monosome). Følgelig kan den translationelle status af et mRNA evalueres ved at overvåge dets association med ribosomer 7.
Denne protokol beskriver isoleringen af polysomer fra seks dage gamle Arabidopsis thaliana kimplanter, den efterfølgende isolering af RNA, og analysen af resultaterne. Polysomes og monosomer er adskilt gennem en sucrosemassefyldegradient. Gradienter opsamles i seks fraktioner. Nogle af de fraktioner samles til opnåelse tre godt adskilt fraktioner: polysomer, monosomer og den lette fraktion (herefter kaldet supernatant), som indeholder de frie 60S og 40S ribosomale subunits og mRNA'er, som ikke er forbundet med ribosomer. Global oversættelse aktivitet kan estimeres ved at frembringe en polysom / monosome forhold, som bestemmes ved integration af arealet under kurven, og ved at sammenligne polysomerne profiler. mRNA'er og proteiner ekstraheres derefter fra de forskellige bestanddele og anvendes til analyse af RT-PCR, QRT-PCR, Northern blot, microarray, western blot eller proteomics. Denne protokol er blevet valideret for andre planter og væv.
Det udstyr, der kræves for at udføre denne protokol er almindeligt forekommende i de fleste laboratorier: Der er ikke behov for en gradient maker. Frysning hvert lag, før du tilføjer den næste forhindres fra enhver blanding eller forstyrrelse af lagene. Nr rør piercer anvendes til gradient samling, som kan opnås ved nedsænkning af et glas kapillarrør i gradienten. Derfor er de kostbare ultracentrifugerør forbliver ubeskadigede og kan genanvendes mange gange. Kollektivt, det gør den nuværende protokol en nem og billig metode til polysom profilering.
Protocol
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af den franske National Research Agency (ANR-14-CE02-0010). Vi takker Dr. Benjamin Field og Dr. Elodie Lanet for kritisk læsning af manuskriptet. Vi takker Mr. Michel Terese for hans hjælp med video redigering.
Materials
| 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 |
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