Este trabalho foi apoiado pela Agência Nacional de Investigação francesa (ANR-14-CE02-0010). Agradecemos Dr. Benjamin Campo e Dr. Elodie Lanet para a leitura crítica do manuscrito. Agradecemos ao Sr. Michel Terese por sua ajuda com edição de vídeo.

1. Preparação de 20 a 50% (p / v) de sacarose Gradientes Nota: Os gradientes são feitos de 4 camadas de sacarose (50%, 35% e 2 camadas de 20%) em 13,2 ml de um tubo de ultracentrífuga. Em nossa experiência, despejando a sacarose 20% em duas camadas separadas melhora muito a qualidade das preparações polissomas. <ol><li> Preparar as soluções de estoque. Certifique-se que todas as soluções são RNAse e DNAse livre. <ol><li> Prepare 10X Solução Salina: Tris-HCl 400 pH 8,4, KCl 200 mM e MgCl 2 100 mM. </li><li> Prepare M de sacarose Solução 2: Para 200 ml, dissolver 137 g de sacarose em 1X solução salina. </li></ol></li><li> Dilui-se a solução de sacarose em solução salina, como descrito na Tabela 1, para preparar os gradientes. Os volumes indicados são para seis gradientes. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Concentração de sacarose final </td><td> sacarose 2H (ml) </td><td> Sal solução 1X (ml) </td><td> Vol final. (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> Tabela 1. As diluições da solução de sacarose para preparar seis gradientes. <ol start="3"><li> Despeje as camadas conforme Tabela 2. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> camada de sacarose </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> Tabela 2. Volume de solução de sacarose por camada. <ol start="4"><li> Depois de verter cada camada, manter os tubos num -40 ° C ou -80 ° C até estar completa congelador de congelação antes da adição de sacarose a camada seguinte. A congelação é geralmente alcançada após 2 h a -40 ° C, mas de esperar cerca de 6 h antes de se verter a camada seguinte é recomendado. A última camada de 20% pode ser congelada ou adicionado de fresco no dia da experiência. Nota: Congelamento cada camada antes de adicionar o próximo impede a partir de qualquer mistura ou perturbação das camadas. Os gradientes podem ser mantidos numa -40 ° C ou -80 ° C congelador durante pelo menos seis meses. </li><li> Se ainda não tiver sido feito, adicionar a última camada de 20% para os gradientes no dia da experiência. Então, deixe-os gradientes descongelar em uma sala fria ou um frigorífico. </li></ol> 2. Preparação de Extratos citossólicos Nota: Nós recomendaçãacabar usando dois gradientes por amostra biológica. 300 mg é a quantidade óptima de material vegetal para preparar dois gradientes quando se trabalha com 6 dias de idade plântulas de Arabidopsis thaliana. Quando se trabalha com tecidos traducionalmente menos activos, a quantidade de material de planta pode ser aumentada até 600 mg. <ol><li> Colheita de seis dias da germinação cultivadas em ½ Murashige e Skoog 8 meio suplementado com 1% de sacarose ou mídia equivalente a-congelação rápida em azoto líquido </li><li> material vegetal moer num almofariz e pilão pré-arrefecida com azoto líquido. </li><li> Pesar 300 mg de material em pó em um prato de pesagem pré-arrefecido. Execute esta etapa rapidamente para evitar o descongelamento da amostra. Nota: Mantenha as amostras congeladas por mais de uma semana em um C congelador -80 °. </li><li> Adicionar 2,4 ml de tampão pré-arrefecida polissoma (Solução Salina 4X, EGTA 5,26 mM, 0,5% (v / v), octilfenoxi poli (etilenoxi) etanol, ramificadas, 50 μg.ml -1 ciclo-heximida, 50 1 μg.mlcloranfenicol) e homogeneizar por mistura com a ponta da pipeta. Transferir 1,5 ml para dois tubos. Trabalhar rapidamente para impedir que as amostras de aquecimento. </li><li> Centrifuga-se a 16000 xg durante 15 min a 4 ° C numa microcentrífuga para sedimentar os detritos. Nota: Para evitar a degradação do RNA, mantenha sempre as amostras a 4 ° C, utilize RNase / DNase soluções livres e trabalhar em RNase / DNase condições livres. A heparina pode ser adicionado ao tampão de polissoma, a uma concentração final de 300 μg.ml -1, para melhorar a protecção de ARN. No entanto, uma vez que a heparina pode interferir com a análise a jusante, que terá de ser removido durante o passo de precipitação de ARN através da realização de precipitação de cloreto de lítio em vez de precipitação com etanol (nota cf. 4.7). </li></ol> 3. polysome Profiling <ol><li> Cuidadosamente pipeta o sobrenadante sem perturbar o sedimento. Se fragmentos de plantas foram pipetados, repita o passo 2.5. Carregar o sobrenadante no topo do gradiente por pipettin suavementeg para a parede lateral do tubo num fluxo constante. Use um gradiente para cada tubo de 1,5 ml. </li><li> Transferência para o pré-arref ecido baldes e centrifugar a 175.000 xg durante 2 h 45 min a 4 ° C, em uma ultracentrífuga (por exemplo 32.000 rpm ao utilizar um rotor SW41). </li><li> Configurar o sistema de recolha de gradiente, tal como descrito na Figura 1. A cuveta UV tem um percurso de 1 mm. </li></ol><img alt="figura 1" src="/files/ftp_upload/54231/54231fig1.jpg" /> Figura 1. Sistema de recolha de inclinação. A cuvete de UV está ligado um tubo de cloreto de polivinilo a um tubo capilar de vidro, que desce para o fundo do gradiente. O gradiente progride através do sistema graças a uma bomba peristáltica. OD 260 é continuamente lido e 2 ml frações são coletadas. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54231/54231fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior</a>esta figura. <ol start="4"><li> Ajustar o colector de fracções para a TA, e definir o carrossel, a uma velocidade que permita a recolha de fracções de 2 mL. Use tubos de coleta pré-arrefecido. </li><li> Recolher as fracções de baixo para cima utilizando um tubo capilar de vidro ligada a um tubo de cloreto de polivinilo para o sistema de recolha de gradiente. Usar um filme de parafina de plástico para assegurar a ligação entre o tubo de cloreto de polivinilo e o tubo capilar de vidro. </li><li> Ler a absorvância a 260 nm a partir do fundo para o topo do gradiente. Quando todo o gradiente é recolhido, coloque os tubos de coleta no gelo antes de extração de RNA. </li></ol> 4. Extracção de ARN <ol><li> Reunir as fracções recolhidas a partir de dois gradientes, em 50 ml tapados tubos de centrifugação, como segue: Polissomas: fracções de 1 a 3 (12 ml) Monosomes: valor correspondente a 4 (4 ml) Sobrenadante: fracções 5 e 6 (8 ml) </li><li> Para cada fracção, adicione 1 vol. Hidrocloreto de guanidina 8M,50 ug transportador acrílico e 1,5 vol. isopropanol. Nota: Acryl transportador é acrilamida linear, usado como um co-precipitante para melhorar a recuperação de ácidos nucleicos durante a precipitação com álcool. </li><li> Misturar por inversão dos tubos e precipitar O / N à temperatura de -20 ° C. </li><li> Centrifugar a 175.000 xg durante 1 h a 4 ° C, em uma ultracentrífuga (32.000 rpm num rotor SW32). Se o passo de precipitação é realizada de um tubo, o qual não é compatível com a ultracentrifugação, transferir para um tubo adequado. </li><li> Elimine o sobrenadante e dissolve-se o sedimento em 200 de tampão TE isento de ARNase ul (Tris-HCl 10 mM, pH 7,5-1 mM de EDTA). Transferir para um novo tubo de 1,5 ml. </li><li> Extrair ARN por adição de 1 vol. fenol saturado de água (pH 6,6) e 1 vol. clorofórmio: álcool isoamílico (24: 1). Misturar energicamente. Centrifuga-se a 15000 xg durante 20 min a 4 ° C. Transferir a fase aquosa para um novo tubo de 1,5 mL. Nota: ácido fenol (pH 4,5) pode ser utilizado a fim de minimizar a contaminação do ADN. </li><li>Precipitar o ARN por adição de 1/10 vol. M de acetato de sódio 3 e 3 vol. 100% de etanol. Permitir a precipitação a -80 ° C durante 20 min ou O / N à temperatura de -20 ° C. Centrifuga-se a 15000 xg durante 15 min a 4 ° C. Nota: Se estiver usando heparina no tampão polysome (nota cf. 2.5), execute um cloreto de lítio precipitação (LiCl) em vez de precipitação de etanol para remover adequadamente qualquer vestígio de heparina que possa interferir com a análise a jusante 9. Após extracção, adicionar LiCl até uma concentração final de 2,5 M. permitir a precipitação durante 30 minutos à temperatura de -20 ° C ou O / N a 4 ° C. Centrifuga-se a 15000 xg durante 15 min a 4 ° C. </li><li> Lavar o sedimento com 500 ul de etanol a 75%. Ar secar o sedimento e dissolvem-se em 30 ul de tampão TE. Avaliar a qualidade do ARN por electroforese num ARNase livre de 1,2% em gel de agarose (100V, 15 min) ou por métodos de electroforese capilar. </li></ol> Análise 5. Os dados <ol><li> Exportação de dados brutos para um software gráfico e de análise de dados. Nota: Como mostrado na Figura 2A e B, os perfis de matérias dar informação qualitativa: o número de picos polissomas que pode ser visto, a altura do pico monosome, e a presença de um ombro correspondente para as subunidades de ribossomas livres. </li><li> Mesclar perfis para permitir a comparação dos perfis entre as amostras. Utilizar a ferramenta de leitor de tela para determinar o valor de X do pico monosome para cada amostra e alinhar os picos monosome removendo pontos de dados adicionais no início das curvas. Identificar o ponto mais baixo da curva e determinar seu valor Y (utilizando a ferramenta de leitor de tela). Defina o valor mais baixo Y ponto a 0 usando a função &quot;valor definido colunas&quot;. </li><li> Determinar a proporção polissomas / monosomes por integração da área sob a curva de perfis em bruto (Figura 3C). Utilizar a ferramenta selector de dados para definir a fronteira da área a ser integrados. Em seguida, use a função de integrar (Análise-Matemática). </li><li> Normalizaras curvas para o pico monosome dividindo todos os pontos de dados de uma curva pelo valor Y da cimeira do pico monosome (determinado utilizando a ferramenta de leitor de tela). Selecione a coluna, então sob análise em Matemática, selecione Normalize e escolher os &quot;dividido por um valor especificado&quot; métodos. Este passo permite a comparação do nível relativo de polissomas (Figura 3D). </li></ol><img alt="Figura 2" src="/files/ftp_upload/54231/54231fig2.jpg" /> Figura 2. Perfis polissomas representativos. Arabidopsis thaliana de tipo selvagem (wt, ecotipo Col-0) e mudas mutantes foram cultivadas durante seis dias em ½ Murashige e Skoog sob fotoperíodos o dia (luz 16 horas, 8 horas escuro). A: perfis polissomas brutos de mudas wt. B: perfis polissomas brutos de mudas mutantes. C: Determinação da percentagem de polissomas e monosomes por integração da área sob a curva D: profi polissomales normalizados para o pico monosome. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54231/54231fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

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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 ribosomes to the mRNA (as cycloheximide does for the cytosolic ribosomes)12 . We have also reduced the total ultracentrifugation time7.
To obtain high quality polysome profiles, it is essential to use freshly collected plant material and to perform all steps at 4&#176;C. When using tissues that are poorly translationally active, more plant material can be loaded on the gradient (up to 600 mg).
Analysis of polysome profiles can provide insights into both the overall translational status of cells13 and the translational status of a specific mRNA. We have used RNAs isolated by this method for different applications. Using the RNAs for microarrays allowed us to identify a class of cadmium stress response genes for which transcription and translation are uncoupled 14. We also used this method to identify small RNAs associated with polysomal fractions. This identification was made by northern blotting15 of RNA extracted from polysomal fractions, and provided biochemical evidence for a translational component in the miRNA pathway in plants. In another study, a cis-NAT RNA was identified by quantitative RT-PCR. This cis-NAT is associated to the phosphate homeostasis and promote translation of the PHO1;2 transcript 16. 
The main limits of the polysome profiling approach are the lack of information concerning both the position of the ribosome on the mRNA and its progression along the mRNA. Ribosome profiling has emerged to address these limitations 17 and has been successfully used on plant tissues18. Ribosome profiling provides a global measurement of translation by taking advantage of the advances in sequencing technology. Nevertheless, as for any sequencing based assays, the quality of the results depends on the mapping of the sequences to the genome, therefore focusing on the small ribosome-protected fragments makes it difficult to deconvolute repetitive sequences. Moreover, the digestion of RNA not protected by the ribosomes leads to the loss of regulatory information contained in the 3' and 5' UTRs. It is then impossible to distinguish transcript variants that have different 3' or 5' UTRs and show different levels of translation19. The polysome profiling method described here is rapid and does not require specific technical skills. Altogether, these two methods represent complementary approaches to study translation regulation.

A síntese de proteínas é um processo essencial e dispendiosa energeticamente em todas as células 1. Em primeiro lugar, as células devem investir energia na produção da maquinaria de tradução, os ribossomas. Por exemplo, uma célula de levedura dividindo ativamente produz tanto quanto 2.000 ribossomas por minuto. Essa produção exige até 60% da actividade de transcrição total e até 90% da actividade total de splicing da célula 2. Além disso, é necessária energia para a síntese de aminoácidos, aminoacil-ARNt e ligações peptídicas. Nas plantas, a adição de um aminoácido a uma cadeia peptídica custos de 4,5 a 5,9 moléculas de ATP 3. Portanto, não é surpreendente que a tradução do mRNA para a proteína é um importante local de regulação, especialmente quando se trata de lidar com a mudança de condições ambientais. A etapa de iniciação da tradução, que é a associação de um mRNA com o ribossoma, é o principal alvo da regulamentação dosTradução 4. Como uma consequência da regulação da tradução, bem como outros passos de regulação pós-transcricional, apenas 40% das variações na concentração de proteína pode ser explicada por ARNm de abundância 5,6. Assim, o estudo de mRNA total fornece relativamente pouca informação sobre a abundância de proteínas. Por outro lado, a associação de mRNA com ribossomos dá melhor visão sobre a abundância de proteínas, dando acesso a esses mRNAs envolvidos na tradução. mRNAs activamente traduzida estão associados com vários ribossomas em estruturas chamadas polissomas. Por outro lado, mRNAs mal traduzidas será associado a apenas um ribossomo (monosome). Por conseguinte, o status translacional de um ARNm pode ser avaliado por monitorização da sua associação com ribossomas 7. Este protocolo descreve o isolamento de polissomas a partir de seis dias de idade plântulas de Arabidopsis thaliana, o subsequente isolamento do ARN e a análise dos resultados. Polysomes monosomes e são separadas através de um gradiente de densidade de sacarose. Gradientes são coletados em seis fracções. Algumas das fracções são reunidas para obter três fracções bem separados: polissomos, monosomes ea fração leve (doravante denominado sobrenadante), que contém os 60 e 40S subunidades ribossomais livres e mRNAs que não estão associados a ribossomas. actividade de tradução global pode ser estimada através da geração de uma relação de polissoma / monosome, que é determinada por integração da área sob a curva, e por comparação dos perfis de polissomas. ARNm e as proteínas são, em seguida, extraiu-se a partir das diferentes fracções e utilizado para análise por RT-PCR, qRT-PCR, Northern blot, microarray, Western blot ou proteómica. Este protocolo foi validado para outras plantas e tecidos. Os equipamentos necessários para realizar este protocolo são comumente encontrados na maioria dos laboratórios: Não há necessidade de uma máquina de inclinação. Congelação cada camada antes de adicionar o próximo prevenirs de qualquer mistura ou perturbação das camadas. Nenhuma perfurador tubo é utilizado para a recolha de inclinação que pode ser obtida por imersão de um tubo capilar de vidro no gradiente. Por conseguinte, os tubos de ultracentrífuga dispendiosos não ficam danificadas e podem ser re-utilizada várias vezes. Coletivamente, isso faz com que o presente protocolo de um método fácil e barato para 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.

Na literatura, os perfis de polissomas são mostrados frequentemente a partir da fracção de luz para a fracção pesada como um resultado da forma como os gradientes são recolhidos, ou seja, a partir do topo para o fundo. Uma vez que no protocolo aqui descrito os gradientes são recolhidos a partir da base para o topo, os perfis que mostram começar com a fração pesada (as polissomos) e vá para a fração de luz (subunidades de ribossomas livres e RNAs) (Figura 2A). Em seguida, recolher cada gradiente em seis fracções de 2 ml, mas fracções de menor dimensão pode ser recolhida se uma análise mais detalhada do conteúdo polissoma tem de ser levada a cabo. Mesclando e normalizando as curvas para o pico monosome (Figura 2D) permite a comparação de perfis de diferentes linhas ou condições de crescimento. Este fornece informação sobre a quantidade relativa de polissomas, independentemente do nível de iniciação. Outra forma de umnalyze os perfis é para calcular a área sob a curva, assim, pode-se determinar a quantidade relativa de ARNm associado tanto com as monosomes ou os polissomas (Figura 2C). Esta relação é específico de uma planta de crescimento e condições. No entanto, esta abordagem pode não ser relevantes no caso de tecidos em translação activo fracamente. Temos utilizado este método para A. plantas inteiras thaliana, rosetas jovens e idosos, bem como para a N. benthamiana (Figura 3C), S. lycopersicum (Figura 3E) e O. folhas de sativa (Figura 3D). A forma do perfil depende das condições de crescimento, idade da planta e os tecidos analisados. Aqui usamos A. thaliana seis dias mudas de idade. Nesta fase, a actividade de translação é alta, e o perfil mostra picos bem perfilada (Figura 2A e B). Este é também o casoquando A. thaliana flores são usadas (Figura 3A). Ao usar 4 semanas de idade A. thaliana rosetas (Figura 3B), as amostras contêm principalmente deixa adulto totalmente desenvolvido, onde as células não se dividem. Assim, o montante global de polissomos e monosomes é menor. O perfil mostra menos, mas ainda bem em forma de picos polissomas. Próximo ao pico monosome, o ombro mostra a grande quantidade de 60S livres subunidades ribossomais. Com outro tipo de plantas ou tecidos, os picos polissomas pode ser pouco visível. Este é o caso quando se usa 30 dias de idade O. folhas de sativa (Figura 3D). Mesmo quando a quantidade de ARNm envolvidos na tradução é muito baixo (nenhum pico polissoma pode ser visto em perfil), a presença do pico monosome indica que o fraccionamento foi adequadamente realizado e que os ARNm pode ser, posteriormente, extraída a partir da fracção para análise posterior . A qualidade da qualidade do RNA extraído é avaliada por electroforese em gel de agarose ( <str<html> ong&gt; Figura 4). O 25S e 18S RNA ribossomal citosólica deve ser claramente visível no gel. Bandas inferior correspondente ao RNA ribossomal cloroplasmático deve também ser visível quando a ARN são extraídos a partir de tecidos verdes 10. <img alt="Figura 3" src="/files/ftp_upload/54231/54231fig3.jpg" /> Figura 3. perfis polysome de diferentes materiais e espécies vegetais. (A) flores Arabidopsis thaliana (300 mg), (B) de Arabidopsis thaliana 4 semanas de idade rosetas (600mg), benthamiana (C) Nicotiana (folhas jovens de 40 dias as plantas cultivadas-dia curto de idade - 300mg), (D) Oryza sativa ( folhas de plantas de 30 dias de idade - 300mg), (E) Solanum lycopersicum (folhas jovens de 35 dias as plantas cultivadas-dia curto de idade - 300mg). Os picos monosome são indicados por setas.iles / ftp_upload / 54231 / 54231fig3large.jpg &quot;target =&quot; _ blank &quot;&gt; Clique aqui para ver uma versão maior desta figura. <img alt="Figura 4" src="/files/ftp_upload/54231/54231fig4.jpg" /> . Figura 4. Avaliação da qualidade do ARN ARN (500 ng extraída a partir de rebentos de 6 dias de idade plântulas de Arabidopsis thaliana) a partir das fracções indicadas foram aplicadas num gel de agarose a 1,2% e separados por electroforese (100V - 15 min). Citosólicas (25S e 18S) rRNA são indicados por setas e cloroplasmático (23S e 16S) rRNA por um suporte. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54231/54231fig4large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

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.

Um método fácil para a planta 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

Nanyang Technological University

Research

JoVE Journal

Biology

12.6K visualizações.  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.

Vídeo: 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.

Polysome de criação de perfil em Leishmania, células humanas e testículo de rato

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 ...

Bioquímica

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.

Dinâmica proteómica e miRNA análise de polissomos de coração de rato isolado após Langendorff perfusão

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.

Perfil de polissomas sem fabricantes de gradientes ou sistemas de fracionamento

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.

Traduzindo a Purificação da Afinidade ribossófica (TRAP) para investigar o desenvolvimento raiz arabidopsis thaliana em uma escala específica de tipo de célula

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

Biologia do Desenvolvimento

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.

Análise de Perfil eucarióticas 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.

Purificação polissóbica de nódulos simbióticos de soja

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.

Análise da Tradução Iniciação Durante condições de estresse por polysome 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.

Polysome Fracionamento e Análise de mamíferos Translatomes em uma escala genômica

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.

Análise da tradução no cérebro do rato em desenvolvimento usando perfil polino

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

Neurociência

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.

Avaliação da Seletiva mRNA Tradução em células de mamíferos por polissoma Profiling

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