Este trabajo fue apoyado por la concesión de la Sinergia de la Fundación de ciencia nacional suizo CRSII3 147697, la Stiftung für Angewandte Krebsforschung y Zürich Krebsliga.

1. preparación de la fracción de proteínas citosólicas y nucleares
<ol><li>Placa 4 x 106 ACC-MESO-4 células en un frasco de cultivo celular T150 cultivadas en RPMI - 1640 medio suplementado con 10% FBS, 1 x penicilina/estreptomicina (100 x) y 2 mM L-glutamina (200 mM). Cuando las células alcanzan una confluencia del 80-90% (~5-5.5 x 106 células), proceder a la extracción de proteínas de la fracción nuclear y citosólica. Nota: Línea de celular de ACC-MESO-4 se obtuvo de la RIKEN BioResource centro11.</li>
	<li>Aspire medio, lave las células añadiendo 15 mL de PBS 1 x, incline la placa suavemente unas cuantas veces y aspirar el PBS 1 x. Añadir 3 mL de 0.25% tripsina-EDTA e Incube el frasco de cultivo a 37 ° C durante 3 minutos. Nota: Observar las células bajo el microscopio para la separación; Si todavía toma el frasco contra la palma de la mano 3 veces.</li>
	<li>Agregar 7 mL de Medio RPMI-1640 suplementado con 10% FBS, penicilina/estreptomicina de 1 x (100 x) y 2 mM L-glutamina (200 mM), recoger las células en un tubo de 15 mL y desactivación a 200 x g durante 5 minutos.</li>
	<li>Medio de aspirar y resuspender el pellet celular con 1,5 mL de 1 x PBS, luego transferir las células a un tubo de microcentrífuga de 1,5 mL. Desactivación a 500 x g y 4 ° C por 5 min, descartar el sobrenadante. Nota: De este paso adelante, realice todos los pasos de centrifugación a 4 ° C y mantener todos los reactivos en el hielo.</li>
	<li>Resuspender el precipitado de células con helado 1,5 mL de 1 x PBS y rotación de las células a 500 x g y 4 ° C por 3 minutos descarte el sobrenadante. Nota: Las cantidades necesarias de CER I, II CER y NER reactivos son 10, 0.55 y 5 volúmenes de pellets Estimado celular lleno de volumen, respectivamente; el siguiente protocolo asume un volumen de 20 μl. Recomendamos preparar únicamente la cantidad necesaria de reactivo CER yo.</li>
	<li>Añadir 200 μL de reactivo de extracción citoplásmico helada (CER I) suplementado con 2 μl de 1 x inhibidor de la proteinasa, por ejemplo, diluir 1 tableta de inhibidor de la proteasa (EDTA-libre) en 500 μl de agua para obtener 100 acciones de x. Esta cantidad de reactivo CER basta para lyse 20 μl de un volumen el sedimento celulares.<ol>
<li>Para estimar el volumen de pellets embalado célula, comparar el tubo que contiene el precipitado de células con un tubo de 1,5 mL con 10, 20, 50 o 100 μl de PBS 1 x.</li>
	</ol>
</li>
	<li>Resuspender el precipitado de células por Vortex vigorosamente el tubo por 15 s e incubar el tubo en hielo por 10 minutos Añadir 11 μl del reactivo de extracción citoplásmico helada II (CER II) al tubo, vortex vigorosamente durante 5 s e incubar 1 minuto en el hielo.</li>
	<li>Vortex vigorosamente durante 5 s y centrifugar el tubo a 16.000 x g y 4 ° C por 5 min transferir el sobrenadante a limpiado tubo previamente enfriado en hielo. El sobrenadante es la fracción citoplasmática que es más utilizado en el experimento de RNA-telecine.</li>
	<li>Resuspender el precipitado quedado en 100 μl de reactivo de extracción nuclear helada (NER) suplementado con 1 μl de inhibidor de la proteinasa (100 x) y vigorosamente en vortex por 15 s. Recomendamos preparar únicamente la cantidad necesaria de reactivo NER suplementado con los inhibidores de la proteasa.</li>
	<li>Incubar la muestra en hielo durante 40 minutos con Vortex ocasional por 15 seg (cada 10 min).</li>
	<li>Centrifugar el tubo a 16.000 x g durante 10 min. traslado el sobrenadante (esto es la fracción de proteína nuclear) a un tubo limpio previamente enfriado.</li>
	<li>Inmediatamente se procederá a medir la concentración de proteínas utilizando el método de bicinchoninic (BCA) (véase Tabla de materiales). Para el control de pureza de proteínas de cada fracción, realizar immunoblotting (figura 1) contra α-tubulina (detección positiva sólo en la fracción citosólica) y de la poli ADP-ribosa polimerasa - PARP (detección positiva sólo en la fracción nuclear). Para la visualización de proteínas, consulte la sección 4.</li>
	<li>Para mantener la actividad de la proteína y su estado natal, preparar 25 μl de alícuotas de extractos citosólicas y nucleares. Rápido-congele estas alícuotas colocándolos en nitrógeno líquido durante 5 s y tienda directamente a-80 ° C.</li>
</ol>2. etiquetado de ARN con Desthiobiotin
<ol><li>Sondas de RNA sintetizado comercialmente y HPLC purificada. Resuspender con agua libre de nucleasas en 10 μm. Nota: Las secuencias sonda se enumeran en la tabla 1.</li>
	<li>Uso 50 pmol de RNA por reacción RNA-telecine. Descongelar y mantener todos los reactivos en el hielo excepto para clavija 30%, utilizado para la etiqueta 3' terminal del RNA oligo.</li>
	<li>5 μl de 10 μm RNA puntas de prueba, etiquetado como CALB2 3' UTR (son) la transferencia, CALB2 3' UTR (mtARE) y ARN-Unrelated (IRE), en 0.5 mL pared delgada microcentrífuga los tubos e incubar en una máquina PCR a 85 ° C por 5 minutos colocar los tubos inmediatamente en hielo. Nota: Este paso es importante, ya que fomenta la relajación y la accesibilidad de la sonda de RNA para el etiquetado.</li>
	<li>Para una sola reacción de 30 μL, añada al tubo que contiene el RNA la siguientes 10 μl mezclar: 3 μl de agua libre de nucleasa, 3 μl de 10 x Buffer de reacción RNA ligasa, 1 μl de inhibidor de la Rnasa (40 U/μL), 1 μl de Desthiobiotinylated citidina bifosfato (1 mM) y 2 μl de ligasa de T4 RNA (20 U/μL). Nota: Para preparar mezcla principal A 3 muestras en exceso (3.2), la mezcla 9,6 μl de nucleasa agua libre, 9,6 μl de 10 x Buffer de reacción RNA ligasa, 3,2 μl de inhibidor de la Rnasa (40 U/μL), 3,2 μl de Desthiobiotinylated citidina bifosfato (1 mM) y 6.4 μL de ligasa de T4 RNA (20 U/μL).</li>
	<li>Agregar cuidadosamente 15 μl de PEG 30% a la reacción. Use otro Consejo para mezclar la reacción. Incubar la reacción durante la noche a 16 ° C.</li>
	<li>Al día siguiente, preparar los siguientes: 5 M de NaCl (fresh nucleasa-libre), cloroformo: alcohol isoamílico en proporción 24:1 (por ejemplo, por reacción - 96 de μl de cloroformo y 4 μL de alcohol isoamílico), helada 100% de etanol y etanol al 70% helada.</li>
	<li>Añadir 70 μl de agua libre de nucleasa en los tubos de la reacción RNA-etiquetado. Añada 100 μl de cloroformo: isoamílico alcohol y agitar brevemente. La desaceleración a 13.000 x g por 3 minutos cuidadosamente quitar sólo la fase superior y la transferencia a un nuevo tubo libre de nucleasa 1.5 mL; Evite tocar la fase inferior.</li>
	<li>Añadir 10 μl de NaCl 5 M, 1 μl de glucógeno y 300 μL de etanol al 100% helado. Coloque el tubo a-20 ° C por 2 h. Nota: En este paso, el experimento puede continuarse al día siguiente.</li>
	<li>Centrifugar a 13.000 x g y 4 ° C durante 15 minutos eliminar cuidadosamente el sobrenadante sin perturbar el pellet. Añadir 300 μL de helada etanol al 70% y centrifugar a 13.000 x g y 4 ° C durante 5 minutos.</li>
	<li>Descartar completamente el sobrenadante y secar el pellet (15 min). Resuspender el precipitado en 20 μl de agua libre de nucleasa. Nota: Proceda con RNA-desplegable en el mismo día.</li>
	<li>Incubar el RNA a 90 ° C por 2 min y coloque en hielo. Coloque el RNA marcado en hielo durante el siguiente paso de prelavado de las bolas magnéticas de estreptavidina. Nota: Un tiempo de incubación más largo puede dañar la sonda de RNA.</li>
</ol>3. ARN-proteína telecine
<ol><li>
Pre-lavado de granos magnéticos de estreptavidina e incubación con desthiobiotin-RNA
	<ol>
<li>Utilizar 50 μl de granos magnéticos estreptavidina por 50 pmol de RNA. Sin embargo, esto debe ser optimizado dependiendo el experimento.</li>
		<li>Vórtice el tubo con granos estreptavidina-magnéticos para 15 s y rápidamente eliminar 200 μl (mezcla principal; suficiente para reacciones de 3 + 1) en un tubos safe-lock de 1,5 mL limpia usando corta puntas de pipeta. Coloque el tubo sobre un soporte magnético para que los granos de recogen en el lado del tubo y esperan 1 minuto, retire el líquido de resuspensión.</li>
		<li>Lavar los granos, quitar el tubo del soporte magnético, añadir 400 μL de 0,1 M NaOH 0.05 M de NaCl solución y suavemente pipeta arriba y abajo varias veces. Coloque el tubo en el soporte magnético. Esperar 1 minuto y recoger el sobrenadante. Repita este paso otra vez.</li>
		<li>Lavar los granos con 200 μL de 100 mM de NaCl. Eliminar el sobrenadante.</li>
		<li>Añadir 200 μL de 20 mM Tris (pH 7.5), suspender cuentas mediante pipeteo, coloque el tubo en el soporte magnético, espere 1 minuto y eliminar el sobrenadante. Repita el paso.</li>
		<li>Retire el tubo del soporte magnético, añadir 200 μL de 1 x de buffer de captura RNA y resuspender granos magnéticos estreptavidina por Vortex brevemente. Quitar 50 μl de granos magnéticos estreptavidina y añada a cada tubo marcado-RNA utilizando una pipeta de corte. Incubar los tubos durante 30 min a temperatura ambiente en un rodillo.</li>
	</ol>
</li>
	<li>
Atascamiento de la proteína al RNA
	<ol>
<li>Colocar los tubos en un soporte magnético, esperar 1 minuto y eliminar el sobrenadante.</li>
		<li>Añadir 50 μl de 20 mM Tris (pH 7.5) a los granos y resuspender mediante pipeteo. Colocar los tubos en un soporte magnético, esperar 1 minuto y eliminar el sobrenadante. Repita este paso.</li>
		<li>Añadir 100 μl de 1 x de tampón de Unión proteína-ARN a las cuentas y resuspender mediante pipeteo.</li>
		<li>Mientras tanto, prepare la siguiente mezcla: 10 μl de 10 x tampón de Unión proteína-RNA, 30 μl de glicerol 50%, 50 μg de proteínas citoplasmáticas y libre de nucleasas hasta 100 μl de agua. Nota: Preparar mezcla master B de 3 muestras en exceso (3.3) como sigue: 33 μl de 10 x tampón de Unión proteína-RNA, 99 μl 50% glicerol, 165 μg de proteínas citoplásmicas y agua libre de nucleasas hasta 330 μl. Mantenga el tubo de mezcla master B en el hielo.</li>
		<li>Colocar los tubos en soporte magnético, espere 1 minuto y recoger el sobrenadante. Añada 100 μl de la mezcla principal B, suspender transfiriendo suavemente hacia arriba y hacia abajo y no crear burbujas. Incubar los tubos de reacción de 60 minutos a 4 ° C en un rodillo.</li>
	</ol>
</li>
	<li>
Lavado y elución
	<ol>
<li>Colocar los tubos en un soporte magnético, recoger el sobrenadante (que es el flujo a través de - FT) y transferir a un tubo nuevo. Nota: Mantener esta FT en hielo para su posterior análisis.</li>
		<li>Pipetear 100 μl de 1 x de tampón de lavado en las cuentas, resuspender suavemente, vuelva a colocar en el soporte magnético, esperar 1 min y descarte el sobrenadante. Repetir este paso 2 veces.</li>
		<li>Agregar 40 μl de tampón de elución a las bolas magnéticas y mezclar mediante pipeteo arriba y abajo, incubar a 37 ° C en un agitador de tubos (950 rpm) durante 30 minutos.</li>
		<li>Colocar los tubos en un soporte magnético, esperar 1 min y recoger muestras eluídas (que es el eluido - E). Coloque en hielo y utilizar para posteriores análisis. Nota: En este paso, cada prueba RNA sonda consiste en un paso (FT) y la muestra de efluente (E).</li>
	</ol>
</li>
</ol>4. poliacrilamida electroforesis y Western Blot análisis
Nota: Consulte la tabla 2 para las recetas de los buffers utilizados en esta sección. Realizar un Western blot según el método de Laemmli12.
<ol><li>Handcast 12% geles de SDS-PAGE (10-pozos, separador de 1,5 mm, 66 μL). Preparar una mezcla de gel de corriente: 4 mL de solución stock de 30% acrilamida-bisacrilamida, 2,4 mL de Tris inferior del almacenador intermediario, 3,2 mL de dH2O, 9,6 μl de TEMED, 96 μl de solución de persulfato de amonio 10% (APS). Preparar la mezcla de gel de apilamiento: 3,25 mL de dH2O 0,5 mL de 30% acrilamida - bisacrilamida solución, 1,3 mL de tampón Tris superior, 10 μl de TEMED, 20 μl de 10% APS.</li>
	<li>Añadir a 16.6 μl de tampón de x Laemmli 6 (p. ej., 14 mL de pH 6.8 de Tris/HCl/SDS (tampón de Tris superior), 2 g de SDS, 1,86 g de la TDT, 6 mL de glicerol y 0.012% bromofenol azul; almacenar a-20 ° C) en la muestra FT y 6,6 μl de tampón de x Laemmli 6 en muestra de E. Incubar las muestras durante 5 min a 95 ° C.</li>
	<li>Las muestras (20 μl del FT y eluir ~ 40 μL) de la carga y correr durante 30 min a 60 V, continuando con 20 V durante la noche.</li>
	<li>Utiliza un sistema de electroblotting semiseco, transferencia de proteínas a una membrana PVDF para 80 min a 15 V. Nota: La membrana PVDF debe ser activada antes de realizar la transferencia de la proteína. Incubar la membrana en metanol al 100% por 1 min, seguido de una incubación de 2 minutos en agua ultrapura.</li>
	<li>Después de la transferencia de la proteína, incubar el gel SDS-PAGE con la solución de Coomassie para 3 h, seguido de tres pasos de lavado con agua ultrapura, cada 30 minutos.</li>
	<li>Al final de la transferencia de la proteína, deje que la membrana PVDF seque por 1 h. reactivar la membrana como se indica en el paso 4.5. Bloquear la membrana 1 h con 5% de BSA en 1 x TTBS.</li>
	<li>Incubar la membrana con el primer anticuerpo: HuR o mesothelin, tanto diluyeron 1: 1000 en 5% de BSA en 1 x TTBS de 4 h a temperatura ambiente o durante la noche a 4 ° C.</li>
	<li>Lavar la membrana tres veces por 7 min en 1 x TTBS e incubar con la peroxidasa conejo anti-ratón (HRP)-anticuerpo secundario conjugado diluido 1: 10,000 en 5% BSA, 1 x TTBS.</li>
	<li>Visualizar las proteínas mediante quimioluminiscencia mejorada y un reproductor de imágenes digital.</li>
</ol>

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Los autores declaran que no tienen intereses financieros que compiten.

3' UTRs pertenecen al genoma no codificante3, y todos los RNAs no codificantes pueden interactuar con proteínas para ejercer su función7. Cuando el genoma mamífero fue encontrado para ser transcrito lo impregna y produjeron una porción significativa de larga noncoding RNAs16, nuevas evidencias demostraron que estos larga noncoding RNAs función en la regulación de expresión génica al interactuar con remodelación de cromatina complejos<sup cla...

Expresión génica y el nivel final del producto del gene pueden ser reguladas fuertemente afectando la estabilidad del ARNm y ARNm traducción tarifa1. Estos mecanismos de regulación postranscripcional se ejercen a través de las interacciones de no codificante del RNA y RNA-que ata las proteínas (restrictivas) con el mRNA específicos. Generalmente es el 3' región sin traducir de mRNA (3' UTR - pertenecientes a la parte no codificante del genoma2) que contiene específico cis-elementos reguladores (CRE), que son reconocidos por trans-actúan factores como miRNA o restrictivas 3. el mejor estudio cis-elemento dentro de los 3' UTR, es el adorno de elementos ricos en adenina (son), que es reconocido por proteínas específicas AU (AUBP) y, a su vez, induce o mRNA degradación/deadenylation (mediada son decaimiento) o mRNA estabilización4.
El tamaño de los 3' UTR del calretinin mRNA (CALB2) es 573 bp largo y contiene una supuesta AUUUA pentámero, como AREsite2, una herramienta bioinformática del5. Consistente con la presencia de un supuesto motivo son, el análisis de vector-reportero de pmirGLO había demostrado un papel de estabilización de este elemento dentro de mRNA CALB26. Por último, el en vitro RNA-telecine se utilizó para identificar la AUBP que estabiliza calretinin mRNA mediante el adorno son.
Pues todo no-codificación RNAs interactúan con proteínas7, el en vitro RNA-telecine es una buena forma y análisis de primera de elección para la identificación de RNA-interactianos a ayudar a descifrar los mecanismos moleculares. En este método de RNA-telecine, comercialmente sintetizadas sondas de RNA, que fueron etiquetadas con una forma modificada de la biotina (desthiobiotin) en la terminal 3' de la hebra de RNA, fueron utilizadas. El RNA-desthiobiotin se inmoviliza mediante la interacción con estreptavidina en granos magnéticos, que se utilizan para tirar abajo proteínas que interaccionan específicamente con el limite-RNA de interés. Proteínas no desnaturalizadas y activas de la fracción citosólica de las células del mesotelioma se utilizan como fuente de proteínas. Tales proteínas RNA-bound se eluyen de las bolas magnéticas, a través de un gel de SDS-PAGE de 12%, transferidos a una membrana y sondeado con los diferentes anticuerpos.
En el procedimiento de purificación de afinidad estándar de estreptavidina-biotina, desnaturalización dura condiciones se requieren interrumpir el vínculo fuerte biotina-estreptavidina irreversible para eluir las proteínas límite8, que podría conducir a la disociación de complejos de la proteína. A diferencia de la biotina, desthiobiotin se une a estreptavidina reversible y se desplaza competitivamente con una solución de biotina, lo que permite la elución suave de proteínas y evitar el aislamiento de naturalmente biotinilado moléculas9 lo que sugiere que la técnica es ideal para el aislamiento de complejos de proteína nativa en condiciones nativas.
In vitro condiciones obligatorias y rigor, que están determinadas por la concentración de sales, agentes reductores y porcentaje de detergente, deben estar cerca de los presentes en el contexto celular con el fin de identificar interacciones de verdad en vivo . Las condiciones vinculantes en este documento han sido previamente demostradas según corresponda para la identificación de lo HuR como una AU-que atan la proteína10. Este enfoque podría ahorrar tiempo, ya que la optimización de las condiciones de enlace adecuada puede ser difícil y desperdiciador de tiempo. Además, este método podría utilizarse como un protocolo inicial para cualquier experimento de RNA-desplegable y puede optimizarse gradualmente cambiando la concentración de sales y detergentes, cambiando el porcentaje de glicerol y adición de otras sales. Por otra parte, hemos demostrado que incluso una corto 25-nucleótido RNA sonda albergando un pentámero son motivo podía utilizarse para demostrar la interacción con una AUBP específico.

<table><tbody><tr><td>NE-PER Nuclear and cytoplasmic extraction kit</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>78833</td><td></td></tr><tr><td>Pierce Protease Inhibitor Tablets, EDTA-free</td><td>Thermo Fisher Scientific</td><td>A32965</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Fisher Scientific</td><td>23225</td><td></td></tr><tr><td>Pierce Magnetic RNA-protein Pull-Down Kit&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>20164</td><td>Includes magnetic beads and therefore the kit need to be stored at 4 &amp;deg;C</td></tr><tr><td>Pierce RNA 3&amp;rsquo; End Desthiobiotinylation Kit&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>20163</td><td>The kit is a part of the &quot;Pierce Magnetic RNA-protein Pull-Down Kit&quot;, and needs to be stored at -20 &amp;deg;C unlike the pull-down kit (4 &amp;deg;C).</td></tr><tr><td>DynaMag-2,&amp;nbsp; magnetic stand</td><td>Thermo Fisher Scientific</td><td>12321D</td><td></td></tr><tr><td>Anti - HuR (MOUSE) monoclonal antibody</td><td>Thermo Fisher Scientific</td><td>-</td><td>The antibody is included in the Pierce Magnetic RNA-protein Pull-Down Kit&amp;nbsp; - 1:1000 in 5% BSA 1x TTBS - rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS</td></tr><tr><td>Anti - &amp;alpha; - tubulin (MOUSE) monoclonal antibody</td><td>Santa Cruz</td><td>8035</td><td>1:1000 in 5% BSA 1x TTBS - rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS</td></tr><tr><td>Anti - PARP (RABBIT) Polyclonal antibody</td><td>Cell Signaling</td><td>9542</td><td>1:1000 in 5% BSA 1x TTBS - goat anti-rabbit secondary antibody 1:10,000 in 5% BSA 1x TTBS</td></tr><tr><td>Anti - Mesothelin (MOUSE) Monoclonal Antibody&amp;nbsp;</td><td>Rockland</td><td>200-301-A87</td><td></td></tr><tr><td>Secondary goat anti-rabbit antibody</td><td>Cell Signaling</td><td>7074</td><td></td></tr><tr><td>Secondary rabbit anti-mouse antibody</td><td>Sigma-Aldrich</td><td>A-9044</td><td></td></tr><tr><td>RPMI - 1640 medium</td><td>Sigma-Aldrich</td><td>R8758</td><td></td></tr><tr><td>FBS - Filtrated Bovine Serum</td><td>Pan Biotech&amp;nbsp;</td><td>P40-37500</td><td></td></tr><tr><td>Penicillin - streptomycin (100x)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P4333</td><td></td></tr><tr><td>L-Glutamine solution (200 mM)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>G7513</td><td></td></tr><tr><td>0.25% Trypsin-EDTA (1x)</td><td>Gibco by Life technologies&amp;nbsp;</td><td>25200-056</td><td></td></tr><tr><td>Mini-PROTEAN 3 Cell</td><td>Bio-Rad</td><td>1653301</td><td></td></tr><tr><td>Mini Protean System Glass Plates&amp;nbsp;</td><td>Bio-Rad</td><td>1653308</td><td></td></tr><tr><td>Mini-PROTEAN Spacer Plates with 1.5 mm integrated spacer</td><td>Bio-Rad</td><td>1653312</td><td></td></tr><tr><td>Mini-PROTEAN Comb, 10-well, 1.5 mm, 66 &amp;micro;L</td><td>Bio-Rad</td><td>1653365</td><td></td></tr><tr><td>Mini-PROTEAN Tetra Cell Casting Modules</td><td>Bio-Rad</td><td>1658050</td><td></td></tr><tr><td>RNaseZap RNase Decontamination Solution</td><td>Thermo Fisher Scientific</td><td>AM9780</td><td></td></tr><tr><td>Rotiphorese gel 30 - aqueous 30% acrylamide and bisacrylamide stock solution at a ration of 37.5:1</td><td>Roth</td><td>3029</td><td></td></tr><tr><td>20% SDS&amp;nbsp;</td><td>PanReac AppliChem</td><td>A3942</td><td></td></tr><tr><td>PVDF transfer membrane&amp;nbsp;</td><td>Perkin Elmer</td><td>NEF1002</td><td>Need to be activated by incubating in pure methanol for 1 min followed by washing in water for 2 min&amp;nbsp;</td></tr><tr><td>Trans-Blot SD semi-dry transfer cell&amp;nbsp;</td><td>Bio-Rad</td><td>1703940</td><td></td></tr><tr><td>Clarity Western ECL Substrate</td><td>Bio-Rad</td><td>1705061</td><td></td></tr><tr><td>FusionFX&amp;nbsp; Digital Imager</td><td>Vilber</td><td>-</td><td></td></tr><tr><td>RNA oligo synthesis</td><td>Mycrosynth AG, Switzerland</td><td>-</td><td>the synthesis scale - 0.04 &amp;micro;mol - HPLC purified</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7030</td><td></td></tr><tr><td>Hydrochloric Acid (HCl) 6 M</td><td>PanReac AppliChem</td><td>182883.1211</td><td></td></tr><tr><td>Ultra Pure 1 M Tris, pH 7.5&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>15567027</td><td></td></tr><tr><td>Glycerol&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>17904</td><td>50% sterile aliquots to be stored at -20&amp;deg;C</td></tr><tr><td>Pierce Streptavidin magnetic beads&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>88817</td><td>Please note that these magnetic beads have an average diameter of 1 &amp;micro;m (range from 0.5 - 1.5 &amp;micro;m). Furthermore, Dynabeads-M280 Streptavidin, which are beads of homogenouse size 2.8 &amp;micro;m, are also used in RNA-pulldown but be aware that this may affect purification process.</td></tr><tr><td>TEMED</td><td>Sigma-Aldrich</td><td>T9281</td><td></td></tr><tr><td>Ammonium persulfate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A3678</td><td></td></tr><tr><td>Coomassie G-250</td><td>Applichem</td><td>A3480</td><td></td></tr><tr><td>Aluminiumsulfate-(14-18)-hydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>368458</td><td></td></tr><tr><td>ortho-phosphoric acid&amp;nbsp;</td><td>Applichem</td><td>A0637</td><td></td></tr></tbody></table>

desthiobiotin-streptavidin-affinity mediated purification of rna-interacting proteins in mesothelioma cells

El en vitro RNA-telecine se utiliza todavía en gran parte en los primeros pasos de protocolos dirigidos a identificar proteínas de unión de ARN que reconocen determinados motivos y estructuras de ARN. En este protocolo de RNA-telecine, comercialmente sintetizadas sondas de RNA están marcadas con una forma modificada de la biotina, desthiobiotin, en el 3' terminal del filamento del RNA, que se une reversiblemente a estreptavidina y permite así la elución de proteínas bajo más fisiológica condiciones. El RNA-desthiobiotin se inmoviliza mediante la interacción con estreptavidina en granos magnéticos, que se utilizan para tirar abajo proteínas que interaccionan específicamente con el RNA de interés. Proteínas no desnaturalizadas y activas de la fracción citosólica de las células del mesotelioma se utilizan como fuente de proteínas. El método aquí descrito puede aplicarse para detectar la interacción entre proteínas RNA conocidas y una sonda de RNA largo (nt) de 25 nucleótidos que contiene una secuencia de interés. Esto es útil para completar la caracterización funcional de estabilizar o desestabilizar elementos presentes en las moléculas de ARN alcanzadas usando un análisis de vectores de reportero.

En este experimento, un fragmento largo 25-nt de calretinin 3' UTR que es motivo (CALB2 3' UTR (son) 25-nt) fue utilizado para probar si se une específicamente a la proteína del antígeno humano R (HuR), un conocido estabilizador de mRNA. Para probar la especificidad del elemento son, un RNA 25 nt sonda CALB2 3' UTR (mtARE) que contiene una mutación son Adorno, que previamente fue demostrada para suprimir el efecto de estabilización del motivo son, fue usado6. ...

Watch this Scientific Journal Video about Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells at JoVE.com

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

Desthiobiotin-estreptavidina-afinidad mediada por purificación de proteínas de interacción con RNA en células de mesotelioma

Desthiobiotin etiquetado de un sintético 25-nucleótido RNA oligo, que contiene un motivo de elementos ricos en adenina (ARE), permite la unión específica de proteína citosólica son.

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA ...

desthiobiotin-streptavidin-affinity-mediated-purification-rna

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

8.0K vistas.  University Hospital Zurich. Desthiobiotin etiquetado de un sintético 25-nucleótido RNA oligo, que contiene un motivo de elementos ricos en adenina (ARE), permite la unión específica de proteína citosólica son.

Video: Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

Nuevas proteínas de unión a ARN aislamiento por la Metodología RaPID

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

Investigación

Genética

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

Desplegable base de biotina de ensayo a validar mRNA miRNAs objetivos de celular

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Saccharomyces cerevisiae Metabólico etiquetado con 4-tiouracilo y la cuantificación de mRNA recién sintetizada como un Proxy para la actividad de ARN polimerasa II

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of polyadenylated mRNAs with antisense oligo(dT) beads and subsequent capture of an mRNA of interest with 3&#39;-biotinylated 2&#39;-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads. TRIP was used to recover in vivo crosslinked mRNA-ribonucleoprotein (mRNP) complexes from yeast, nematodes and human cells for further RNA and protein analysis. Thus, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to study the dynamic re-arrangement of mRNPs imposed by intracellular or environmental cues.

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

Un procedimiento de aislamiento de RNA tándem basada en oligonucleótidos recuperar complejos eucariotas ARNm-proteína

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein ...

Bioquímica

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

Ensayos bioquímicos in Vitro utilizando etiquetas de biotina para estudiar las interacciones entre proteínas y ácidos nucleicos

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial purification of biologically important RNA/protein complexes. However, this strategy suffers from one major disadvantage that limits its broader utilization: the biotin/streptavidin interaction can be broken only under denaturing conditions that also disrupt the integrity of the eluted complexes, hence precluding their subsequent functional analysis and/or further purification by other methods. In addition, the eluted samples are frequently contaminated with the background proteins that nonspecifically associate with streptavidin beads, complicating the analysis of the purified complexes by silver staining and mass spectrometry. To overcome these limitations, we developed a variant of the biotin/streptavidin strategy in which biotin is attached to an RNA substrate via a photo-cleavable linker and the complexes immobilized on streptavidin beads are selectively eluted to solution in a native form by long wave UV, leaving the background proteins on the beads. Shorter RNA binding substrates can be synthesized chemically with biotin and the photo-cleavable linker covalently attached to the 5&#39; end of the RNA, whereas longer RNA substrates can be provided with the two groups by a complementary oligonucleotide. These two variants of the UV-elution method were tested for purification of the U7 snRNP-dependent processing complexes that cleave histone pre-mRNAs at the 3&#39; end and they both proved to compare favorably to other previously developed purification methods. The UV-eluted samples contained readily detectable amounts of the U7 snRNP that was free of major protein contaminants and suitable for direct analysis by mass spectrometry and functional assays. The described method can be readily adapted for purification of other RNA binding complexes and used in conjunction with single- and double-stranded DNA binding sites to purify DNA-specific proteins and macromolecular complexes.

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

Solo paso purificación de complejos macromoleculares utilizando RNA unido a biotina y un enlazador foto escindibles

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial ...

Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate regulation of these processes. A strategy called CLIP-seq (cross-linking and immunoprecipitation) has been developed to capture endogenous protein-RNA interactions with UV cross-linking followed by immunoprecipitation. Despite the wide use of conventional CLIP-seq method in RBP study, the CLIP method is limited by the availability of high-quality antibodies, potential contaminants from the copurified RBPs, requirement of isotope manipulation, and potential loss of information during a tedious experimental procedure. Here we describe a modified CLIP-seq method called FbioCLIP-seq using the FLAG-biotin tag tandem purification. Through tandem purification and stringent wash conditions, almost all the interacting RNA-binding proteins are removed. Thus, the RNAs interacting indirectly mediated by these copurified RBPs are also reduced. Our FbioCLIP-seq method allows efficient detection of direct protein-bound RNAs without SDS-PAGE and membrane transfer procedures in an isotope-free and protein-specific antibody-free manner.

Here we present a modified CLIP-seq protocol called FbioCLIP-seq with FLAG-biotin tandem purification to determine the RNA targets of RNA-binding proteins (RBPs) in mammalian cells.

Perfilado en todo el transcriptoma de las interacciones entre proteínas y ARN por enlace cruzado e inmunoprecipitación mediada por la purificación en tándem FLAG-Biotin

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate ...

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying the binding partners of an RNA of interest remains of high importance to unveil the mechanisms behind many cellular processes. However, RNA molecules might interact transiently and dynamically with some RNA-binding proteins (RBPs), especially with non-canonical ones. Hence, improved methods to isolate and identify such RBPs are greatly needed.
To identify the protein partners of a known RNA sequence efficiently and quantitatively, we developed a method based on the pull-down and characterization of all interacting proteins, starting from cellular total protein extract. We optimized the protein pull-down using biotinylated RNA pre-loaded on streptavidin-coated beads. As a proof of concept, we employed a short RNA sequence known to bind the neurodegeneration-associated protein TDP-43 and a negative control of a different nucleotide composition but the same length. After blocking the beads with yeast tRNA, we loaded the biotinylated RNA sequences on the streptavidin beads and incubated them with the total protein extract from HEK 293T cells. After incubation and several washing steps to remove nonspecific binders, we eluted the interacting proteins with a high-salt solution, compatible with the most commonly used protein quantification reagents and with sample preparation for mass spectrometry. We quantified the enrichment of TDP-43 in the pull-down performed with the known RNA binder compared to the negative control by mass spectrometry. We used the same technique to verify the selective interactions of other proteins computationally predicted to be unique binders of our RNA of interest or of the control. Finally, we validated the protocol by western blot via the detection of TDP-43 with an appropriate antibody. 
This protocol will allow the study of the protein partners of an RNA of interest in near-to-physiological conditions, helping uncover unique and unpredicted protein-RNA interactions.

Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis.

Un análisis extraíble cuantitativo optimizado de proteínas de unión al ARN utilizando ARN biotinilado corto

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying ...

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination1-7. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism8-13. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.
Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA14. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotin-containing messages.
The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,14-16. To examine the function of these isolated ribosome complexes, peptidyl-transferase assays can be performed in the presence of the antibiotic puromycin1. To study structural changes in translational complexes, well established procedures can be used, such as i) crosslinking to specific amino acids14 and/or ii) alkylation protection assays1,14,17.

A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.

El aislamiento de los ribosomas traducir contienen peptidil-ARNt para el Análisis Funcional y Estructural

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

Biología

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

La detección de las proteínas de unión a ARN por<em&gt; In Vitro</em&gt; ARN desplegable de adipocitos Cultura

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...