Questo lavoro è stato sostenuto dall&#39;Agenzia Nazionale della Ricerca francese (ANR-14-CE02-0010). Ringraziamo il dottor Benjamin campo e il Dr. Elodie Lanet per la lettura critica del manoscritto. Ringraziamo il Sig Michel Terese per il suo aiuto con il video editing.

1. Preparazione del 20 al 50% (w / v) di saccarosio gradienti Nota: I gradienti sono fatti di strati 4 di saccarosio (50%, 35% e 2 strati di 20%) in un tubo ultracentrifuga 13,2 ml. Nella nostra esperienza, versando il saccarosio del 20% in due strati separati migliora notevolmente la qualità delle preparazioni polysome. <ol><li> Preparare le soluzioni madre. Assicurarsi che tutte le soluzioni sono RNAsi e DNAsi gratuito. <ol><li> Preparare 10X soluzione salina: 400 mm Tris-HCl pH 8.4, 200 mm e 100 mm KCl MgCl 2. </li><li> Preparare 2 M saccarosio Soluzione: Per 200 ml, sciogliere 137 g di saccarosio in 1X soluzione salina. </li></ol></li><li> Diluire la soluzione di saccarosio in soluzione salina, come descritto nella Tabella 1, per preparare i gradienti. I volumi indicati sono per sei pendenze. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> La concentrazione di saccarosio finale </td><td> saccarosio 2M (ml) </td><td> Sale soluzione 1X (ml) </td><td> Vol finale. (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> Tabella 1. Le diluizioni della soluzione di saccarosio per preparare sei gradienti. <ol start="3"><li> Versare gli strati come da Tabella 2. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> strato di saccarosio </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> Tabella 2. Volume di soluzione di saccarosio per strato. <ol start="4"><li> Dopo aver versato ogni strato, tenere i tubi in un -40 ° C o -80 ° C fino a completa freezer congelamento prima di aggiungere il successivo strato di saccarosio. Il congelamento viene solitamente raggiunta dopo 2 ore a -40 ° C, ma si consiglia di attendere circa 6 ore prima di versare lo strato successivo. L&#39;ultimo strato 20% può essere congelato o aggiunta di fresco il giorno dell&#39;esperimento. Nota: congelamento ogni strato prima di aggiungere il successivo impedisce qualsiasi mescolanza o disturbo degli strati. Gradienti possono essere conservati in una -40 ° C o -80 ° C freezer per almeno sei mesi. </li><li> Se non è già stato fatto, aggiungere l&#39;ultimo strato 20% ai gradienti il ​​giorno dell&#39;esperimento. Poi, lasciare che i gradienti scongelare in una stanza fredda o di un frigorifero. </li></ol> 2. Preparazione di citosoliche Estratti Nota: Raccomfinire utilizzando due gradienti per campione biologico. 300 mg è la quantità ottimale di materiale vegetale per preparare due gradienti quando si lavora con 6 giorni di età piantine Arabidopsis thaliana. Quando si lavora con tessuti meno traduzionale attivi, la quantità di materiale vegetale può essere aumentata fino a 600 mg. <ol><li> Harvest sei giorni vecchie piantine coltivate in ½ Murashige e Skoog 8 di media integrato con 1% di saccarosio o mezzi equivalenti dal rapido congelamento in azoto liquido </li><li> materiale vegetale macinare in un mortaio e pestello pre-raffreddata con azoto liquido. </li><li> Pesare 300 mg di materiale in polvere in un piatto di pesatura preraffreddato. Eseguire questo passaggio rapidamente per evitare lo scongelamento del campione. Nota: Mantenere i campioni congelati per non più di una settimana in un congelatore C -80 °. </li><li> Aggiungere 2,4 ml di tampone polysome pre-raffreddata (Salt Solution 4X, 5,26 mM EGTA, 0,5% (v / v) Octylphenoxy poli (etilenossi) etanolo, ramificati, 50 μg.ml -1 cicloesimide, 50 μg.ml 1cloramfenicolo) e omogeneizzare mescolando con la punta della pipetta. Trasferire in due provette da 1,5 ml. Lavorare velocemente per evitare che i campioni da riscaldamento. </li><li> Centrifugare a 16.000 xg per 15 min a 4 ° C in una microcentrifuga a pellet detriti. Nota: per evitare la degradazione dell&#39;RNA, tenere sempre i campioni a 4 ° C, usare soluzioni libere RNasi / DNasi e il lavoro in condizioni di libero RNasi / DNasi. Eparina può essere aggiunto al buffer polysome, ad una concentrazione finale di 300 μg.ml -1, per migliorare la protezione RNA. Tuttavia, poiché l&#39;eparina può interferire con l&#39;analisi valle, dovrà essere rimosso durante la fase di precipitazione RNA eseguendo litio precipitazione cloruro invece di precipitazione con etanolo (cfr nota 4.7). </li></ol> 3. polysome Profiling <ol><li> pipetta con attenzione il surnatante senza disturbare il pellet. Se i frammenti vegetali sono stati pipettati, ripetere il punto 2.5. Caricare il surnatante sopra del gradiente da pipettin delicatamenteg sulla parete laterale del tubo in un flusso costante. Utilizzare una pendenza per ogni provetta da 1,5 ml. </li><li> Trasferimento preraffreddato secchi e centrifugare a 175.000 xg per 2 ore 45 min a 4 ° C, in un&#39;ultracentrifuga (es 32.000 rpm utilizzando un rotore SW41). </li><li> Impostare il sistema di raccolta gradiente come descritto nella figura 1. La cuvetta UV ha un cammino ottico 1 mm. </li></ol><img alt="Figura 1" src="/files/ftp_upload/54231/54231fig1.jpg" /> Figura 1. sistema di raccolta Gradiente. La cuvetta UV è collegato da polivinil cloruro di tubo ad un tubo capillare di vetro che scende verso il fondo del gradiente. Il gradiente procede attraverso il sistema attraverso una pompa peristaltica. OD 260 è continuamente letto e 2 ml frazioni vengono raccolte. <a href="https://www.jove.com/files/ftp_upload/54231/54231fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande</a>questa figura. <ol start="4"><li> Regolare il raccoglitore di frazioni di RT, e impostare il carosello ad una velocità che permetterà la raccolta di 2 ml frazioni. Utilizzare tubi pre-raffreddato di raccolta. </li><li> Raccogliere le frazioni dal basso verso l&#39;alto utilizzando un tubo capillare di vetro collegato da tubi di cloruro di polivinile per il sistema di raccolta gradiente. Utilizzare una pellicola di paraffina in plastica per assicurare il collegamento tra il tubo di cloruro di polivinile e il tubo capillare di vetro. </li><li> Leggere l&#39;assorbanza a 260 nm dal basso verso l&#39;alto del gradiente. Quando l&#39;intero gradiente viene raccolto, posizionare i tubi di raccolta sul ghiaccio prima estrazione di RNA. </li></ol> 4. RNA Estrazione <ol><li> Pool frazioni raccolte da due gradienti, in 50 ml con tappo provette da centrifuga, come segue: Polisomi: frazioni da 1 a 3 (12 ml) Monosomes: frazione 4 (4 ml) Surnatante: frazioni 5 e 6 (8 ml) </li><li> Per ogni frazione, aggiungere 1 vol. 8M guanidina cloridrato,50 mcg vettore acrilico e 1,5 vol. isopropanolo. Nota: Acrilico vettore è acrilammide lineare, usato come coprecipitant per migliorare il recupero di acidi nucleici durante la precipitazione alcool. </li><li> Miscelare invertendo i tubi e precipitare O / N a -20 ° C. </li><li> Centrifugare a 175.000 xg per 1 ora a 4 ° C, in un&#39;ultracentrifuga (32.000 rpm in un rotore SW32). Se la fase di precipitazione viene eseguita in un tubo che non è compatibile con ultracentrifugazione, trasferire ad un tubo adatto. </li><li> Gettare il surnatante e sciogliere il precipitato in 200 microlitri di buffer TE RNasi-free (10 mM Tris-HCl pH 7,5-1 mM EDTA). Trasferire in una nuova provetta da 1,5 ml. </li><li> Estratto di RNA con l&#39;aggiunta di 1 vol. acqua fenolo saturo (pH 6,6) e 1 vol. cloroformio: alcool isoamilico (24: 1). Mescolare energicamente. Centrifugare a 15.000 xg per 20 min a 4 ° C. Trasferire la fase acquosa in una nuova provetta da 1,5 ml. Nota: fenolo acido (pH 4.5) può essere utilizzato per ridurre al minimo la contaminazione del DNA. </li><li>Precipitato RNA con l&#39;aggiunta di 1/10 vol. 3 M acetato di sodio e 3 vol. 100% di etanolo. Consentire precipitazioni a -80 ° C per 20 minuti o O / N a -20 ° C. Centrifugare a 15.000 xg per 15 min a 4 ° C. Nota: Se si usa eparina nel buffer polysome (cfr nota 2.5), eseguire un cloruro di litio (LiCl) la precipitazione invece di precipitazione in etanolo per rimuovere correttamente ogni traccia di eparina che possa interferire con l&#39;analisi a valle 9. Dopo l&#39;estrazione, aggiungere LiCl ad una concentrazione finale di 2,5 M. Lasciar precipitazione per 30 minuti a -20 ° C o O / N a 4 ° C. Centrifugare a 15.000 xg per 15 min a 4 ° C. </li><li> Lavare il pellet con 500 microlitri 75% di etanolo. Far asciugare il pellet e sciogliere in 30 microlitri di buffer TE. Valutare la qualità di RNA mediante elettroforesi su un RNasi gratis 1,2% gel (100V, 15 min) o con metodi elettroforesi capillare. </li></ol> Analisi 5. I dati <ol><li> Esportazione dati grezzi ad un software grafico e l&#39;analisi dei dati. Nota: Come mostrato in Figura 2A e B, i profili grezzi forniscono informazioni qualitative: il numero di picchi polysome che può essere visto, l&#39;altezza del picco monosome, e la presenza di una spalla corrispondente alle subunità ribosomi liberi. </li><li> Unire i profili per permettere il confronto dei profili tra i campioni. Utilizzare lo strumento lettore di schermo per determinare il valore X del picco monosome per ogni campione e allineare i picchi monosome rimuovendo punti dati supplementari all&#39;inizio delle curve. Identificare il punto più basso della curva e determinare il valore Y (utilizzando lo strumento di lettore di schermo). Impostare il valore Y punto più basso a 0 utilizzando la funzione &quot;Imposta valore colonne&quot;. </li><li> Determinare il rapporto polisomi / monosomes integrando l&#39;area sotto la curva di profili grezzi (Figura 3C). Utilizzare lo strumento di selezione dati per definire il bordo dell&#39;area da integrare. Quindi utilizzare la funzione di integrare (Analisi-matematica). </li><li> Normalizzarele curve a picco monosome dividendo tutti i punti dati di una curva per il valore Y del vertice del picco monosome (determinati utilizzando lo strumento di lettura dello schermo). Selezionare la colonna, poi sotto analisi-matematica, selezionare Normalizza e scegliere le &quot;divise da un valore specificato&quot; metodi. Questo passaggio consente il confronto del livello relativo di polisomi (Figura 3D). </li></ol><img alt="figura 2" src="/files/ftp_upload/54231/54231fig2.jpg" /> Figura 2. Profili polysome rappresentativi. Arabidopsis thaliana wild-type (WT, ecotipo Col-0) e piantine mutanti sono state coltivate per sei giorni in ½ Murashige e medie Skoog sotto fotoperiodi giorno lunghe (luce 16 ore, 8 ore scure). A: profili polysome prime provenienti da piante WT. B: profili polysome prime provenienti da piante mutanti. C: Determinazione della percentuale di polisomi e monosomes dalla integrazione dell&#39;area sotto la curva D: profi polysomeles normalizzata al picco monosome. <a href="https://www.jove.com/files/ftp_upload/54231/54231fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a>

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</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...

La sintesi proteica è un processo essenziale e energeticamente costosa in tutte le cellule 1. Prima di tutto, le cellule devono investire energie nella produzione delle macchine di traduzione, i ribosomi. Per esempio una cellula di lievito che divide attivamente produce fino a 2.000 ribosomi al minuto. Tale produzione richiede fino al 60% del totale attività trascrizionale e fino al 90% dell&#39;attività splicing totale della cella 2. Inoltre, l&#39;energia è necessaria per la sintesi di amminoacidi,-tRNA e legami peptidici. Nelle piante, l&#39;aggiunta di un aminoacido ad un costo catena peptidica da 4,5 a 5,9 molecole di ATP 3. Pertanto, non è sorprendente che la traduzione di mRNA di proteina è un importante sito di regolazione, in particolare quando si tratta di trattare con mutevoli condizioni ambientali. Il passo inizio della traduzione, che è l&#39;associazione di un mRNA con il ribosoma, è l&#39;obiettivo principale del regolamento ditraduzione 4. Come conseguenza della regolazione della traduzione nonché altri passaggi normativi post-trascrizionale, solo il 40% delle variazioni della concentrazione di proteine ​​può essere spiegato con mRNA abbondanza 5,6. Così, lo studio di mRNA totale fornisce informazioni relativamente scarsa su l&#39;abbondanza di proteine. D&#39;altra parte, l&#39;associazione di mRNA con ribosomi dà una migliore comprensione abbondanza di proteine, dando accesso a tali mRNA coinvolti nella traduzione. mRNA attivamente tradotti sono associati a diversi ribosomi in strutture chiamate polisomi. Al contrario, mRNA mal tradotti saranno associati con una sola ribosoma (monosome). Di conseguenza, lo stato traduzionale di mRNA può essere valutata monitorando la sua associazione con ribosomi 7. Questo protocollo descrive l&#39;isolamento di polisomi da sei giorni piantine Arabidopsis thaliana, il successivo isolamento di RNA, e l&#39;analisi dei risultati. Polysomes e monosomes sono separati attraverso un gradiente di densità di saccarosio. Le sfumature sono raccolti in sei frazioni. Alcune delle frazioni viene riunito per ottenere tre frazioni ben separati: polisomi, monosomes e la frazione leggera (di seguito denominato surnatante), che contiene le libere 60 e 40S subunità ribosomiale e mRNA che non sono associati con i ribosomi. attività di traduzione globale può essere stimato generando un rapporto segnale / rumore monosome polysome, che è determinata dalla integrazione dell&#39;area sotto la curva, e confrontando i profili polisomi. mRNA e le proteine ​​vengono poi estratte da diverse frazioni e utilizzati per l&#39;analisi mediante RT-PCR, qRT-PCR, Northern blot, microarray, macchia occidentale o la proteomica. Questo protocollo è stato convalidato per altre piante e tessuti. Il materiale necessario per effettuare questo protocollo si trovano comunemente nella maggior parte dei laboratori: Non vi è alcuna necessità di un creatore di gradiente. Congelamento ogni strato prima di aggiungere il successivo prevenires da qualsiasi miscela o disturbo degli strati. N perforatore tubo è utilizzato per la raccolta di gradiente che può essere raggiunto mediante immersione di un tubo capillare di vetro nel gradiente. Pertanto, i tubi ultracentrifuga costose rimangono integre e possono essere riutilizzati più volte. Collettivamente, questo rende il presente protocollo un metodo semplice ed economico per il profiling polysome.

<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 letteratura, profili polysome sono spesso mostrati dalla frazione leggera alla frazione pesante come una conseguenza del modo gradienti vengono raccolti, cioè dalla parte superiore alla parte inferiore. Poiché nel protocollo descritto qui le pendenze sono raccolti dal basso verso l&#39;alto, i profili mostriamo iniziano con la frazione pesante (i polisomi) e andare alla frazione leggera (subunità libera ribosoma e RNA) (Figura 2A). Abbiamo poi raccogliere ogni sfum...

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.

Un metodo facile per piante 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 ...

an-easy-method-for-plant-polysome-profiling

Research

JoVE Journal

Biology

12.5K 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

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.

Polisoma Profiling nel testicolo di topo, cellule umane e di Leishmania

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 ...

Ricerca

Biochimica

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.

Dinamica proteomica e miRNA analisi di polisomi da Mouse cuore dopo Langendorff aspersione isolata

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.

Profilazione dei polisomi senza generatori di gradienti o sistemi di frazionamento

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

Tradurre la purificazione dell'affinità del ribosoma (TRAP) per studiare lo sviluppo della radice di Arabidopsis thaliana su una scala specifica del tipo di cella

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

Biologia dello sviluppo

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.

Eucarioti Analisi Profilo Polyribosome

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

Biologia

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.

Purificazione dei polisomi dai noduli simbiotici di soia

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

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.

Analisi della traduzione innesco durante condizioni di stress da polisoma Profiling

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.

Polisoma Frazionamento e analisi dei mammiferi Translatomes su scala Genome-wide

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.

Analisi della traduzione nel cervello del mouse in via di sviluppo utilizzando la profilazione polisome

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

Neuroscienze

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.

Valutazione della Selective mRNA Traduzione in cellule di mammifero per polisoma Profiling

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