Name: an easy method for plant polysome profiling
Uploaded: 2016-08-28T14:00:00.000+00:00
Duration: 11 min 9 s
Description: Watch this Scientific Journal Video about An Easy Method for Plant Polysome Profiling at JoVE.com

Este trabajo fue apoyado por la Agencia Nacional de Investigación (ANR-14-CE02-0010). Se agradece al Dr. Benjamin campo y el Dr. Elodie Lanet para la lectura crítica del manuscrito. Agradecemos al Sr. Michel Terese por su ayuda con la edición de vídeo.

1. Preparación de 20 a 50% (p / v) de sacarosa gradientes Nota: Los gradientes son de 4 capas de sacarosa (50%, 35% y el 2 capas de 20%) en un tubo de ultracentrífuga 13.2 ml. En nuestra experiencia, el vertido de la sacarosa al 20% en dos capas separadas mejora en gran medida la calidad de las preparaciones polisomas. <ol><li> Preparar las soluciones madre. Asegúrese de que todas las soluciones son de RNAsa y DNAsa. <ol><li> Preparar 10X Solución Salina: mM Tris-HCl pH 8,4 400, KCl 200 mM y MgCl 100 mM 2. </li><li> Preparar la solución 2 M de sacarosa: Por 200 ml, se disuelven 137 g de sacarosa en 1X solución salina. </li></ol></li><li> Diluir la solución de sacarosa en Salt Solution, como se describe en la Tabla 1, para preparar los gradientes. Los volúmenes indicados se refieren a seis gradientes. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> La concentración de sacarosa final </td><td> sacarosa 2M (ml) </td><td> La solución salina que 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> Tabla 1. Las diluciones de la solución de sacarosa para preparar seis gradientes. <ol start="3"><li> Verter las capas según la Tabla 2. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> capa de sacarosa </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> Tabla 2. Volumen de la solución de sacarosa por capa. <ol start="4"><li> Después de verter cada capa, mantener los tubos en un -40 ° C o -80 ° C congelador hasta congelación completa antes de añadir la siguiente capa de sacarosa. La congelación se consigue normalmente después de 2 horas a -40 ° C, pero esperando sobre 6 hr antes de verter la siguiente capa se recomienda. La última capa 20% se puede congelar o añadido recientemente en el día del experimento. Nota: Congelación cada capa antes de añadir el siguiente impide cualquier mezcla o perturbación de las capas. Los gradientes se pueden mantener en un -40 ° C o -80 ° C congelador durante al menos seis meses. </li><li> Si ya no se ha hecho, añadir la última capa 20% a los gradientes en el día del experimento. A continuación, dejar que los gradientes se descongelan en una habitación fría o una nevera. </li></ol> 2. Preparación de extractos citosólicos Nota: Se recomterminar utilizando dos gradientes por muestra biológica. 300 mg es la cantidad óptima de material vegetal para preparar dos gradientes cuando se trabaja con 6 días plántulas de Arabidopsis thaliana. Cuando se trabaja con tejidos menos translationally activos, la cantidad de material de la planta se puede incrementar hasta 600 mg. <ol><li> Cosecha de seis días de edad plántulas cultivadas en ½ Murashige y Skoog 8 medio suplementado con 1% de sacarosa o medios equivalentes por congelación rápida en nitrógeno líquido </li><li> material vegetal molido en un mortero y una maja previamente enfriada con nitrógeno líquido. </li><li> Pesar 300 mg de material en polvo en un plato de pesada previamente enfriado. Realice este paso rápidamente para evitar la descongelación de la muestra. Nota: Mantenga las muestras congeladas por no más de una semana en un congelador a -80 °. </li><li> Añadir 2,4 ml de tampón de polisomas preenfriada (sal Solución 4X, EGTA 5,26 mM, 0,5% (v / v) octilfenoxi poli (etilenoxi) etanol, ramificados, 50 ìg.mL -1 cicloheximida, 50 ìg.mL 1cloranfenicol) y homogeneizar mediante la mezcla con la punta de la pipeta. Transferir 1,5 ml en dos tubos. Trabajar con rapidez para evitar que las muestras de calentamiento. </li><li> Centrifugar a 16.000 xg durante 15 min a 4 ° C en una microcentrífuga para sedimentar los desechos. Nota: Para evitar la degradación del ARN, mantener siempre las muestras a 4 ° C, utilizar las soluciones y de trabajo libre de RNasa / DNasa en RNasa / DNasa condiciones libres. La heparina puede ser añadido a la memoria intermedia de polisomas, a una concentración final de 300 ìg.mL -1, para mejorar la protección de ARN. Sin embargo, ya que la heparina puede interferir con análisis de aguas abajo, que tendrá que ser retirado durante la etapa de precipitación del ARN mediante la realización de la precipitación de cloruro de litio en lugar de la precipitación con etanol (cf. nota 4,7). </li></ol> 3. Perfiles de polisomas <ol><li> Con cuidado la pipeta el sobrenadante sin perturbar el sedimento. Si los fragmentos de plantas han sido con pipeta, repita el paso 2.5. Cargar el sobrenadante en la parte superior del gradiente por pipettin suavementeg sobre la pared lateral del tubo en una corriente constante. Utilice uno de gradiente para cada tubo de 1,5 ml. </li><li> Transferencia a preenfriado cubos y se centrifuga a 175.000 xg durante 2 hr 45 min a 4 ° C, en una ultracentrífuga (por ejemplo 32.000 rpm utilizando un rotor SW41). </li><li> Configurar el sistema de recolección de gradiente tal como se describe en la Figura 1. La cubeta UV tiene una longitud de trayectoria de 1 mm. </li></ol><img alt="Figura 1" src="/files/ftp_upload/54231/54231fig1.jpg" /> Figura 1. sistema de recogida de gradiente. La cubeta UV está conectado mediante un tubo de cloruro de polivinilo a un tubo capilar de vidrio que desciende hasta el fondo del gradiente. El gradiente avanza en el sistema gracias a una bomba peristáltica. DO 260 es leído de forma continua y se recogen fracciones de 2 ml. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54231/54231fig1large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de</a>esta figura. <ol start="4"><li> Ajuste el colector de muestras hasta la temperatura ambiente, y establecer el carrusel a una velocidad que permita la recogida de fracciones de 2 ml. Use tubos pre-enfriado de recogida. </li><li> Reunir las fracciones de abajo hacia arriba usando un tubo capilar de vidrio conectada mediante un tubo de cloruro de polivinilo al sistema de recolección de gradiente. Utilizar una película de parafina de plástico para asegurar la conexión entre el tubo de cloruro de polivinilo y el tubo capilar de vidrio. </li><li> Leer la absorbancia a 260 nm de la parte inferior a la parte superior del gradiente. Cuando se recoge todo el degradado, colocar los tubos de recogida en el hielo antes de la extracción de RNA. </li></ol> 4. La extracción de RNA <ol><li> Reunir las fracciones recogidas a partir de dos gradientes, en tapón de 50 ml tubos de centrífuga, como sigue: Polisomas: fracciones 1 a 3 (12 ml) Monosomas: fracción 4 (4 ml) De sobrenadante: fracciones 5 y 6 (8 ml) </li><li> Para cada fracción, se añade 1 vol. hidrocloruro de guanidina 8M,50 g de soporte acrílicas y de 1,5 vol. isopropanol. Nota: portador Acryl es acrilamida lineal, que se utiliza como un coprecipitante de mejorar la recuperación de ácidos nucleicos durante la precipitación de alcohol. </li><li> Mezclar invirtiendo los tubos y precipitar O / N a -20 ° C. </li><li> Centrifugar a 175.000 xg durante 1 hora a 4 ° C, en una ultracentrífuga (32.000 rpm en un rotor SW32). Si la etapa de precipitación se lleva a cabo en un tubo que no es compatible con la ultracentrifugación, transferir a un tubo adecuado. </li><li> Desechar el sobrenadante y disolver el precipitado en 200 l de tampón TE libre de ARNasa (Tris-HCl 10 mM pH 7,5 a 1 mM de EDTA). Transferir a un nuevo tubo de 1,5 ml. </li><li> Extracto de ARN mediante la adición de 1 vol. fenol saturado de agua (pH 6,6) y 1 vol. cloroformo: alcohol isoamílico (24: 1). Mezclar vigorosamente. Centrifugar a 15.000 xg durante 20 min a 4 ° C. Transferir la fase acuosa a un nuevo tubo de 1,5 ml. Nota: fenol ácido (pH 4.5) se puede utilizar con el fin de minimizar la contaminación de ADN. </li><li>Precipitar el ARN mediante la adición de un décimo vol. acetato de sodio 3 M y 3 vol. 100% de etanol. Permitir la precipitación a -80 ° C durante 20 minutos o O / N a -20 ° C. Centrifugar a 15.000 xg durante 15 min a 4 ° C. Nota: Si se utiliza heparina en el tampón de polisomas (véase la nota 2.5), realizar una precipitación de cloruro de litio (LiCl) en lugar de la precipitación con etanol para eliminar adecuadamente cualquier rastro de heparina que pueda interferir con el análisis de aguas abajo 9. Después de la extracción, añadir LiCl a una concentración final de 2,5 M. permitir la precipitación durante 30 min a -20 ° C o O / N a 4 ° C. Centrifugar a 15.000 xg durante 15 min a 4 ° C. </li><li> Lavar el precipitado con 500 l 75% de etanol. Deje secar al aire el precipitado y se disuelve en 30 l de tampón TE. Evaluar la calidad del RNA por electroforesis en un gel de agarosa libre de 1,2% de ARNasa (100 V, 15 min) o por métodos de electroforesis capilar. </li></ol> Análisis 5. Datos <ol><li> Exportar datos en bruto a un software gráfico y análisis de datos. Nota: Como se muestra en la Figura 2A y B, los perfiles primas dan información cualitativa: el número de picos polisomas que se puede ver, la altura del pico de monosome, y la presencia de un hombro correspondiente a las subunidades de ribosomas libres. </li><li> Combinar perfiles para permitir la comparación de los perfiles entre las muestras. Utilice la herramienta de lector de pantalla para determinar el valor X del pico monosome para cada muestra y alinear los picos monosome mediante la eliminación de puntos de datos adicionales en el inicio de las curvas. Identificar el punto más bajo de la curva y determinar su valor de Y (con la función de lector de pantalla). Establecer el valor de Y punto más bajo a 0 mediante el uso de la función &quot;valor de columnas del conjunto&quot;. </li><li> Determine la relación polisomas / monosomas integrando el área bajo la curva a partir de perfiles en bruto (Figura 3C). Utilice la herramienta de selección de datos para definir la frontera de la zona a integrarse. A continuación, utilice la función de integrar (Análisis-Matemáticas). </li><li> Normalizarlas curvas a la cima monosome dividiendo todos los puntos de datos de una curva por el valor Y de la cumbre del pico monosome (determinaron utilizando la herramienta de lector de pantalla). Seleccione la columna, a continuación, en Análisis-Matemáticas, seleccione Normalizar y elegir las &quot;dividido por un valor especificado&quot; métodos. Este paso permite la comparación del nivel relativo de polisomas (Figura 3D). </li></ol><img alt="Figura 2" src="/files/ftp_upload/54231/54231fig2.jpg" /> Figura 2. Perfiles de polisomas representativos. Arabidopsis thaliana de tipo silvestre (wt, ecotipo Col-0) y plántulas mutantes se cultivaron durante seis días en ½ Murashige y Skoog bajo fotoperiodos el día (16 horas de luz, 8 horas de oscuridad). R: perfiles de polisomas primas procedentes de las plantas de semillero en peso. B: Los perfiles de polisomas primas de plántulas mutantes. C: Determinación del porcentaje de polisomas y monosomas por integración de la área bajo la curva D: Profi polisomasles normalizó al pico monosome. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54231/54231fig2large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta 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.

La síntesis de proteínas es un proceso esencial y energéticamente costoso en todas las células 1. En primer lugar, las células deben invertir energía en la producción de la maquinaria de traducción, los ribosomas. Por ejemplo, una célula de levadura dividiendo activamente produce tanto como 2000 ribosomas por minuto. Una producción de este tipo requiere de hasta 60% de la actividad transcripcional total y hasta 90% de la actividad total de empalme de la célula 2. Además, se requiere energía para la síntesis de aminoácidos, aminoacil-tRNA y enlaces peptídicos. En las plantas, la adición de un aminoácido a un costos de la cadena de péptido de 4,5 a 5,9 moléculas de ATP 3. Por lo tanto, no es sorprendente que la traducción de ARNm de la proteína es un sitio importante de la regulación, en particular cuando se trata de lidiar con el cambio de las condiciones ambientales. La etapa de iniciación de la traducción, que es la asociación de un ARNm con el ribosoma, es el principal objetivo de la regulación deTraducción 4. Como consecuencia de la regulación de la traducción, así como otras medidas de regulación post-transcripcional, sólo el 40% de las variaciones en la concentración de proteína se puede explicar por la abundancia de ARNm 5,6. Por lo tanto, el estudio de ARNm total de da relativamente pobre información sobre la abundancia de proteínas. Por otra parte, la asociación de ARNm con ribosomas da una mejor penetración en la abundancia de proteínas, dando acceso a esos mRNAs implicados en la traducción. mRNAs traducidos de forma activa se asocian con varios ribosomas en estructuras llamadas polisomas. Por el contrario, los ARNm mal traducidas estarán asociadas con un solo ribosoma (monosome). En consecuencia, el estado de la traducción de un ARNm puede ser evaluada mediante el control de su asociación con ribosomas 7. Este protocolo describe el aislamiento de polisomas procedentes de seis días plántulas de Arabidopsis thaliana, el posterior aislamiento de RNA, y el análisis de los resultados. Correoslysomes y monosomas se separan a través de un gradiente de densidad de sacarosa. Los gradientes se recogen en seis fracciones. Algunas de las fracciones se agrupan para obtener tres fracciones separadas así: polisomas, monosomas y la fracción ligera (de aquí en adelante llamado sobrenadante), que contiene los libres 60S y 40S ribosomal subunidades y mRNAs que no están asociados con los ribosomas. actividad de traducción Global se puede estimar mediante la generación de un / relación de polisomas monosome, que se determina por la integración del área bajo la curva, y mediante la comparación de los perfiles de polisomas. mRNAs y proteínas se extraen a partir de las diferentes fracciones y se utilizaron para el análisis por RT-PCR, QRT-PCR, transferencia de Northern, microarray, Western blot o la proteómica. Este protocolo ha sido validado para otras plantas y tejidos. El equipo necesario para llevar a cabo este protocolo se encuentran comúnmente en la mayoría de los laboratorios: No hay necesidad de un fabricante de gradiente. Congelación de cada capa antes de añadir el siguiente prevenirs de cualquier mezcla o perturbación de las capas. No perforador tubo se utiliza para la recogida de gradiente que puede lograrse mediante la inmersión de un tubo capilar de vidrio en el gradiente. Por lo tanto, los tubos de ultracentrífuga costosos permanecen sin daños y se pueden volver a utilizar muchas veces. En conjunto, esto hace que el presente protocolo de un método sencillo y barato para los perfiles de polisomas.

<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.

En la literatura, los perfiles de polisomas a menudo se muestran a partir de la fracción ligera de la fracción pesada como resultado de la forma en que se recogen los gradientes, es decir, desde la parte superior a la parte inferior. Dado que en el protocolo descrito aquí los gradientes son recogidos desde el fondo hasta la parte superior, los perfiles mostrados comienzan con la fracción pesada (los polisomas) e ir a la fracción ligera (subunidades de ribosomas libres y ARN) (Figura 2A). A continuación recogemos cada gradiente en seis fracciones de 2 ml, pero fracciones más pequeñas se pueden recoger si un análisis más detallado del contenido de polisomas tiene que ser llevado a cabo. La fusión y la normalización de las curvas a la cima monosome (Figura 2D) permite la comparación de los perfiles de diferentes líneas o las condiciones de crecimiento. Esto proporciona información sobre la cantidad relativa de polisomas independientemente del nivel de iniciación. Otra forma de unanalyze los perfiles es calcular el área bajo la curva, por lo tanto, se puede determinar la cantidad relativa de mRNA asociado con cualquiera de los monosomas o los polisomas (Figura 2C). Esta relación es específico de una planta y condiciones de crecimiento. Sin embargo, este enfoque puede no ser relevante en el caso de los tejidos en traslación activo poco. Hemos utilizado este método para A. plantas enteras thaliana, rosetas jóvenes y viejos, así como para N. benthamiana (Figura 3C), S. lycopersicum (Figura 3E) y O. hojas sativa (Figura 3D). La forma del perfil depende de las condiciones de crecimiento, edad de la planta y los tejidos analizados. Aquí hemos utilizado A. thaliana seis días las plantas de semillero de edad. En esta etapa, la actividad de traducción es alta, y el perfil de muestra así picos de forma (Figura 2A y B). Este es también el casocuando A. thaliana flores se utilizan (Figura 3A). Cuando se utilizan 4 semanas de edad A. thaliana rosetas (Figura 3B), las muestras contienen principalmente adulto plenamente desarrollado hojas donde las células no se dividen. Por lo tanto, la cantidad total de polisomas y monosomas es menor. El perfil muestra un menor número, pero aún así en forma de picos polisomas. Al lado del pico monosome, el hombro muestra la gran cantidad de 60S libres subunidades ribosomales. Con otro tipo de plantas o tejidos, los picos de polisomas pueden ser apenas visible. Este es el caso cuando se utiliza 30 días de edad O. hojas sativa (Figura 3D). Incluso cuando la cantidad de ARNm implicados en la traducción es muy baja (sin pico polisomas se puede ver en el perfil), la presencia del pico monosome indica que el fraccionamiento se realiza correctamente y que los ARNm se pueden extraer más lejos de la fracción para su posterior análisis . La calidad de la calidad del ARN extraído se evaluó por electroforesis en gel de agarosa ( <str<html> ong&gt; Figura 4). El 25S y 18S ARN ribosomal citosólica debe ser claramente visible en el gel. Bandas inferiores correspondientes al ARN ribosomal cloroplástico también deben ser visibles cuando el ARN se extrae de los tejidos verdes 10. <img alt="figura 3" src="/files/ftp_upload/54231/54231fig3.jpg" /> Figura 3. Los perfiles de polisomas de diferente material vegetal y especies. (A) las flores Arabidopsis thaliana (300 mg), (B) de Arabidopsis thaliana 4 semanas de edad rosetas (600 mg), benthamiana (C) Nicotiana (hojas jóvenes de 40 días de edad plantas día de cosecha cortos - 300 mg), (D) Oryza sativa ( hojas de plantas de 30 días de antigüedad - 300mg), (E) Solanum lycopersicum (hojas jóvenes de 35 días de edad plantas de día corto-crecido - 300 mg). Los picos monosome se indican mediante flechas.iles / ftp_upload / 54231 / 54231fig3large.jpg &quot;target =&quot; _ blank &quot;&gt; Haga clic aquí para ver una versión más grande de esta figura. <img alt="Figura 4" src="/files/ftp_upload/54231/54231fig4.jpg" /> . Figura 4. Evaluación de la calidad del ARN ARN (500 ng extrajeron a partir de brotes de 6 días plántulas de Arabidopsis thaliana) de las fracciones indicadas se cargaron en un gel de agarosa al 1,2% y separadas por electroforesis (100V - 15 min). Citosólicas (25S y 18S rRNA) se indican mediante puntas de flecha y los cloroplastos (23S y 16S rRNA) por un soporte. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54231/54231fig4large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta 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.

Un método fácil para la planta de perfiles de polisomas

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

Universidad San Sebastian

Research

JoVE Journal

Biology

12.5K vistas.  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

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

ARNm Interactome Captura de Planta Protoplastos

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Investigación

Bioquímica

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 perfilado en Leishmania, las células humanas y testículo de ratón

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 ...

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.

Proteómicos y miRNA análisis de polisomas de corazón de ratón aislados después de Langendorff la perfusión

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.

Perfilado de polisomas sin fabricantes de gradiente ni sistemas de fraccionamiento

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

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.

Análisis eucariotas perfil 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 ...

Biología

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.

Purificación de polisomas a partir 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 ...

Biología del Desarrollo

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.

Análisis de la Traducción Iniciación Durante condiciones de estrés por 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 Fraccionamiento y análisis de los mamíferos Translatomes en un genoma de gran escala

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.

Análisis de la traducción en el cerebro del ratón en desarrollo utilizando perfiles de polisoma

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

Neurociencias

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.

Evaluación del selectivo ARNm traducción en células de mamífero por polisoma perfiles

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