Este trabajo fue financiado por Singapur NRF comunión (NRF-2011NRF-NRFF001-025), así como CBRG (NMRC / CBRG / 0070/2014).

NOTA: El ARN de interés en el contexto de este estudio es un fragmento de AR 3&#39;UTR. 1. Preparación de ARN marcado con biotina <ol><li> Para obtener el ARN, amplificar T7-AR-oligo por PCR, seguido por la transcripción in vitro utilizando un kit comercial y siguiendo las instrucciones del fabricante 11. NOTA: Los cebadores para PCR se enumeran en la Tabla 4: T7-AR-F y T7-AR-R. </li><li> Para el control de la unión no específica, amplificar una secuencia parcial del ADN de luciferasa de luciérnaga (FL) mediante PCR a partir psiCHECK-2 plásmido, seguido por la transcripción in vitro. NOTA: Los cebadores para PCR se enumeran en la Tabla 4: hluc (+) - T7-sonda-F y hluc (+) - sonda-R Tabla 4 Secuencias de molde y los cebadores para la amplificación por PCR... </li><li> Para un 50 l de reacción de PCR, mezclar 50 ng de ADN (oligo o plásmido), 4 dNTPs l (2,5 mM de cada dNTP individuales), 5 l de 10x tampón de reacción, 1 l 2,5 U / &amp; #181; l de ADN polimerasa, 2 l 10 mM de cebador directo, 2 l 10 mM cebador inverso, y agua libre de nucleasa. <ol><li> Ejecutar la amplificación por PCR en un termociclador usando el siguiente programa. Paso 1: un ciclo de 95 ° C durante 2 min; Paso 2: 35 ciclos de 95 ° C durante 30 segundos, 55 ° C durante 30 segundos y 72 ° C durante 30 segundos (AR) o 60 segundos (FL); Paso 3: un ciclo de 72 ° C durante 10 min. </li></ol></li><li> Sintetizar ARN con biotina utilizando un kit de transcripción in vitro siguiendo las instrucciones del fabricante 11. En este experimento, utilizar productos de PCR como plantillas para la síntesis de ARN. Para una reacción de 20 l transcripción in vitro, mezclar 200 ng de ADN amplificado por PCR, 2 l de cada NTP individual, 1 l 10 mM de biotina-CTP, tampón de reacción de 2 l de 10x, y 2 l de ARN polimerasa de T7. Incubar la mezcla a 37 ° C durante 4 h. </li><li> Después de la transcripción in vitro, mezcla de reacción tratamiento con 1 l 2 U de DNasa / l a 37 &amp; #176; C durante 15 minutos para degradar la plantilla de ADN. Por último, se diluye la ARN biotinilado a un volumen total de 60 l para la purificación posterior. </li><li> Se purifica ARN con biotina para eliminar las sales y los nucleótidos no incorporados mediante el uso de columnas de purificación siguiendo las instrucciones del fabricante 12. Medir la concentración de ARN y se almacena a -80 ° C durante pull-down ensayos posteriores. </li><li> Para asegurar la estructura secundaria apropiada para el ARN de interés por la interacción de ARN-proteína, de calor 50 l de ARN biotinilado (50 pM) a 90 ° C durante 2 min, enfriar en hielo durante 2 min, a continuación, suministrar con 50 l de 2x tampón de la estructura del ARN (Tabla 3), y permitir el plegamiento del ARN a TA durante 30 min 13,14. </li></ol> 2. Preparación de perlas de ARN conjugado NOTA: Utilice un soporte magnético para la separación de las perlas magnéticas y sobrenadante. <ol><li> Volver a suspender las perlas magnéticas recubiertas con estreptavidina en el vial original mediante una breve vortexing siguiendo las instrucciones del fabricante. Transferir 150 l perlas se volvieron a suspender a un tubo de 1,5 ml. Se coloca el tubo en el imán durante 1 min. sobrenadante de descarte. Mantenga sedimento de perlas. NOTA: En los ensayos de pull-down de ARN, cuando una alícuota de perlas se volvieron a suspender a partir vial original, utilizar 50 perlas l para un ensayo seguido por el Western Blot, y 200 bolas l para un ensayo seguido de la EM. </li><li> Lavar perlas una vez por la re-suspensión de sedimento de perlas con 1 ml de recomendada por el fabricante 1x W &amp; B tampón (Tabla 3) (unión y lavado), seguido de girar el tubo durante 5 minutos a temperatura ambiente en un rotador. sobrenadante de descarte. Mantenga sedimento de perlas. </li><li> Para inactivar la RNasa sobre perlas, perlas de lavar dos veces, cada vez por la re-suspensión de sedimento de perlas con 400 l de tampón A. El tampón A es una solución alcalina que contiene NaOH 0,1 M y 0,05 M NaCl (Tabla 3). Girar el tubo durante 2 min a temperatura ambiente en un rotador. sobrenadante de descarte. Mantenga sedimento de perlas. </li> &lt;li&gt; Para eliminar NaOH a partir de perlas, perlas de lavar dos veces, cada vez por la re-suspensión de sedimento de perlas con 400 l de tampón B (Tabla 3), seguido por la rotación del tubo durante 2 minutos a temperatura ambiente en un rotador. sobrenadante de descarte. Mantenga sedimento de perlas. </li><li> Volver a suspender sedimento de perlas con 300 l 2 x W &amp; B tampón, perlas dividida en partes iguales y transferir en tres tubos de 1,5 ml (100 granos l por tubo). Suministrar los tubos que contienen de talones con 100 l de RNasa libre de agua, 100 l con biotina ARN FL y 100 l biotina AR 3&#39;UTR ARN, respectivamente. </li><li> Incubar cada mezcla de microesferas durante 1 hr a temperatura ambiente con rotación. Colocar los tubos en el imán durante 1 min. Desechar todo el sobrenadante. Mantener los gránulos de talón. </li><li> Lavar perlas dos veces, cada vez por la re-suspensión de cada sedimento de perlas con 400 l 1x W &amp; B tampón, seguido de tubos giratorio durante 5 min a temperatura ambiente en un rotador. </li><li> Colocar los tubos en el imán durante 1 min. Desechar todo el sobrenadante. Resuspender cada grano pellet con 50 l de tampón de aislamiento nuclear (Tabla 3). </li></ol> 3. Aislamiento de preadipocitos y adipogénesis inducción <ol><li> Como se ha descrito en publicaciones anteriores 5, diseccionar preadipocitos de la almohadilla de grasa marrón interescapular (C57BL6 ratones de tipo salvaje, 3 semanas de edad). </li><li> Crecer y diferenciarse in vitro los preadipocitos 5. Las células están listas para la aplicación aguas abajo 4-5 días después de la iniciación de la diferenciación. </li></ol> 4. Preparación de lisado celular de los adipocitos primarios NOTA: Asegúrese de que todos los procedimientos de preparación de lisado celular se realizan en el hielo, y todos los tampones se enfrió en hielo. Pre-térmica del homogeneizadores de vidrio Dounce en hielo. <ol><li> Crecer y diferenciarse preadipocitos en 15 cm platos 5 (ver sección 3.2). Cada plato proporciona células adecuadas para el ensayo desplegable uno ARN. En el día 4 o 5 días de diferenciación, medio de cultivo celular de descarte, y ce lavarLLS una vez con PBS 1x. </li><li> Para cosechar las células, añadir 10 a 15 ml de 1x PBS a las células, raspar para recoger las células en un tubo de 50 ml, y las células de pellets por centrifugación a 200 xg durante 5 min a TA. Desechar el sobrenadante mediante el uso de 1 ml pipeta. </li><li> Resuspender pellet de células en 2 ml de tampón hipotónico (Tabla 3), e incubar las células en hielo durante 15 min. La transferencia de células a un homogeneizador de vidrio Dounce de 15 ml, y las células de cizalla mecánicamente en hielo con 20 golpes. </li><li> Pellets núcleos de las células por centrifugación a 3.300 xg durante 15 min a 4 ° C. Mantenga pellets núcleos en hielo para su uso posterior (ver sección 4.6). Transferir el sobrenadante a tubos de 1,5 ml, añadir 3M KCl al sobrenadante para obtener una concentración final de 150 mM, y centrifugado a 20.000 xg durante 15 min a 4 ° C. </li><li> Después de la centrifugación, retirar con cuidado lipidlayeron la parte superior mediante el uso de una pipeta de 1 ml, y recoger el sobrenadante como el lisado celular citoplasmática. </li><li> Resuspender pellet núcleos (ver sección 4.4) en 1 ml de tampón de aislamiento nuclear. Tnúcleos RANSFERENCIA a un homogeneizador de vidrio Dounce de 15 ml usando una pipeta de 1 ml, y los núcleos de cizalla mecánica sobre hielo con 100 golpes. Sedimentar los restos nucleares por centrifugación a 20.000 xg durante 15 min a 4 ° C, y recoger el sobrenadante como el lisado celular nuclear. </li><li> O bien combinar los lisados ​​celulares nucleares y citoplasmáticos, o utilizar cada fracción por separado, para su aplicación desplegable aguas abajo. En este experimento de demostración, estas dos fracciones de lisado de células se combinaron a una relación en volumen de 1: 1. </li><li> Añadir inhibidor de RNasa a este lisado celular no fraccionada para obtener una concentración final de 0,5 U / l. Destinará 100 l de lisado celular como control de entrada para el Western Blot 15. </li></ol> 5. encuadernación y elución de RBP <ol><li> lisado de células de Split en tres alícuotas iguales (lisado de células de 1 ml en un tubo de 1,5 ml), e incubar con blanco, FL RNA-conjugado y perlas de AR 3&#39;UTR ARN-conjugados (véase la sección 2.8), respectivamente, durante 3 horas a 4 ° docon la rotación. </li><li> Colocar los tubos en el imán durante 1 min. Recoger el sobrenadante usando una pipeta de 1 ml, seguido de aislamiento de ARN a partir del sobrenadante para confirmar RNA integralidad. Aislar el ARN mediante el uso de una solución de tiocianato de guanidinio-fenol ácido siguiendo las instrucciones del fabricante (Tabla 2). Evaluar la integridad de ARN mediante el análisis de 28S y 18S subunidades de ARN ribosómico. Mantener los gránulos de bolas en hielo. </li><li> Lavar las perlas magnéticas seis veces, cada vez por resuspensión cada sedimento de perlas con 1 ml de tampón de aislamiento nuclear que contiene 40 U de RNasa inhibidor y 0,25% de Igepal, seguido de tubos giratorio para 2 min a temperatura ambiente en un rotador. Utilice un soporte magnético para la separación de las perlas magnéticas y sobrenadante. Desechar todo el sobrenadante. Mantener los gránulos de bolas en hielo. </li><li> Utilice los pasos siguientes para eluir los complejos de ARN-proteína de los granos para el Western Blot. En primer lugar, volver a suspender cada sedimento de perlas en 75 l de tampón de aislamiento nuclear; a continuación, se añaden 25 l de 4x western blot carga buffer (que contiene SDS) para obtener un volumen total de 100 l. Finalmente, hervir perlas en esta solución 100 l a 95 ° C durante 5 min. NOTA: Utilice los siguientes pasos para eluir los complejos de ARN-proteína de los granos para la EM. Paso 1: resuspender cada sedimento de perlas en 100 l de tampón de elución (Tabla 3); PASO 2: incubar muestras de perlas durante 2-3 horas a temperatura ambiente con rotación; PASO 3: centrífuga y recoger el sobrenadante que contiene proteína; PASO 4: concentrar soluciones de proteínas de 20-30 l antes de su presentación para la EM. </li><li> Centrifugar cada 100 l de muestra de perlas a 12.000 xg durante 30 segundos a 4 ° C, y recoger el líquido sobrenadante. Alícuota de sobrenadante de 15 l para el análisis de western blot (ver sección 6.1 y 6.2). Sonda de la membrana resultante con el anticuerpo monoclonal Hur ratón diluido (dilución 1: 1000), durante la noche. </li></ol> 6. Transferencia Western para la verificación de las prácticas comerciales restrictivas <ol><li> Las prácticas comerciales restrictivas separadas por SDS-PAGE utilizando técnicas estándar. </li><li> realizar nospopa secante utilizando técnicas estándar mediante el sondeo de las prácticas comerciales restrictivas asociados con el ARN de interés. NOTA: En este experimento de demostración, se utilizó el anticuerpo monoclonal de ratón Hur para la inmunodetección de la proteína Hur. </li><li> Incubar la membrana resultante con el anticuerpo Hur diluido (dilución 1: 1000) en 5% w / v de leche en polvo sin grasa, 1x TBS, 0,1% Tween - 20 al 4 ° C con agitación suave, durante la noche. Lavar la membrana tres veces con 1x TBST. Incubar la membrana con el anticuerpo secundario (de cabra anti-IgG de ratón-HRP) a temperatura ambiente durante 1 hr. membrana Wash. Desarrollar y señal de grabación. </li></ol>

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No hay conflictos de interés declarado.

lncRNAs y las prácticas comerciales restrictivas tienen un papel vital en la salud y la enfermedad, sin embargo los mecanismos moleculares de estas moléculas son poco conocidos. Identificación de proteínas que interactúan con moléculas lncRNA es un paso clave para dilucidar los mecanismos de regulación. El enriquecimiento de las prácticas comerciales restrictivas por el sistema de ensayo desplegable ARN se basa en la captura en solución de interactuar complejo de manera que las proteínas de un lisado de célul...

A correction to the author list was made to: <a href="http://www.jove.com/video/54207/detection-rna-binding-proteins-vitro-rna-pull-down-adipocyte">Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture</a>.
The author list has been updated from:
Qianfan Bai1*, Zhiqiang Bai1*, Lei Sun1,2
1Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School
2Institute of Molecular and Cell Biology, Singapore
*These authors contributed equally
to:
Qianfan Bai1*, Zhiqiang Bai1*, Shaohai Xu3, Lei Sun1,2
1Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School
2Institute of Molecular and Cell Biology, Singapore
3Division of Bioengineering, School of Chemical &#38; Biomedical Engineering, Nanyang Technological University
*These authors contributed equally

A correction to the author list was made to: Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture.

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

Prácticas comerciales restrictivas son proteínas que se unen al ARN de doble o de cadena sencilla en las células y participan en la formación de complejos de ARN-proteína. Las prácticas comerciales restrictivas pueden unir una variedad de especies de ARN, incluyendo ARN no codificantes de ARNm y largos (lncRNAs), y ejercer su influencia a nivel post-transcripcional. Ambas prácticas comerciales restrictivas y lncRNAs están surgiendo como nuevos reguladores en el desarrollo adiposo y funcionan 1,2,3. Para entender el mecanismo de RBP- y regulación lncRNA mediada de vías celulares, a menudo es necesario para detectar la interacción entre una molécula de ARN específico y una o varias prácticas comerciales restrictivas, y, a veces, para identificar el espectro completo de proteínas asociados de un ARN transcripción. Sin embargo, el experimento puede ser difícil debido al alto contenido de lípidos en los adipocitos. El protocolo desplegable RNA descrito aquí se puede emplear para recuperar los compañeros de la proteína de un ARN específico de los lisados ​​de adipocitos de cultivos primarios 4,5. Justificación de este protoCol se resume de la siguiente manera. Un cebo de ARN es transcrito in vitro a partir de un molde de ADN, marcado con biotina y conjugado a perlas magnéticas recubiertas con estreptavidina 4. El cebo de ARN se incuba con lisados ​​celulares para permitir la formación de complejos de ARN-proteína, que se extraen posteriormente hacia abajo en un soporte magnético. Más específicamente, el protocolo desplegable RNA describe a continuación utilizar un fragmento de ARN de AR 3&#39;UTR como el cebo para recuperar proteína Hur, un RBP expresado universalmente unido a 3&#39;UTR de ARNm de 6,7, de theadipocytelysate de cultivo primario. Para probar la especificidad de unión de este ensayo, un RNAas no relevantes así como perlas de estreptavidina en blanco se incluyen como control. Este protocolo es compatible con el Western Blot o espectrometría de masas (MS) para confirmar la captura de un RBP específica o para identificar el repertorio completo de las prácticas comerciales restrictivas capturados 8, respectivamente. Hay disponibles varias técnicas para estudiar la interacción proteína-ARNs. Para revelar los ARN unidos por un determinado RBP, RIP (ARN inmunoprecipitación) y CLIP (reticulación UV e inmunoprecipitación) se puede aplicar. Por el contrario, para identificar proteínas asociados de un determinado ARN, ARN desplegable, Chirp (cromatina aislamiento mediante la purificación del ARN), gráfico (análisis de hibridación de captura de dianas de ARN) y el PAR (purificación de ARN antisentido) se puede aplicar. En comparación con los posteriores, la técnica desplegable ARN requiere menos esfuerzo de configurar. Puede ser empleado para capturar proteínas in vitro e in vivo. El enfoque in vivo de ARN desplegable, que es técnicamente más difícil que su homólogo in vitro, preserva las interacciones ARN-proteína por reticulación en las células, capta los ARN aptamer etiquetado de interés a partir de células, y, posteriormente, detecta las prácticas comerciales restrictivas consolidados. ARN desplegable se puede utilizar para enriquecer las prácticas comerciales restrictivas bajas abundantes, y para aislar e identificar los complejos de proteínas y ARN que tienen diversos roles funcionales en el control de la regulación celular 9,10 </sarriba&gt;.

<table><tbody><tr><td>&lt;strong&gt;Major materials used in this study.&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MEGAscript kit</td><td>&amp;nbsp;Ambion</td><td></td><td></td></tr><tr><td>Biotin-14-CTP&amp;nbsp;</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>NucAway spin columns&amp;nbsp;</td><td>Ambion</td><td></td><td></td></tr><tr><td>Dynabeads M-280 Streptavidin&amp;nbsp;</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>HuR (3A2) mouse monoclonal antibody (sc-5261)&amp;nbsp;</td><td>Santa Cruz Biotechnology</td><td></td><td></td></tr><tr><td>Goat anti-mouse IgG-HRP(sc-2005)&amp;nbsp;</td><td>Santa Cruz Biotechnology</td><td></td><td></td></tr><tr><td>Hypure Molecular Biology Grade Water (nuclease-free)&amp;nbsp;</td><td>HyClone</td><td></td><td></td></tr><tr><td>Phosphate Buffer Saline (1&amp;times;PBS)</td><td>&amp;nbsp;HyClone</td><td></td><td></td></tr><tr><td>RNase inhibitor&amp;nbsp;</td><td>Bioline</td><td></td><td></td></tr><tr><td>Protease inhibitor&amp;nbsp;</td><td>Sigma</td><td></td><td></td></tr><tr><td>Pfu Turbo DNA polymerase&amp;nbsp;</td><td>Agilent Technologies</td><td></td><td></td></tr><tr><td>TRIsure</td><td>&amp;nbsp;Bioline</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Major equipment used in this study.&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sorvall Legend Micro 21R centrifuge&amp;nbsp;</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Sorvall Legend X1R centrifuge&amp;nbsp;</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Bio-Rad T100 thermal cycler&amp;nbsp;</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Nanodrop 2000&amp;nbsp;</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>BOECO rotator Multi Bio RS-24&amp;nbsp;</td><td>BOECO,Germany</td><td></td><td></td></tr><tr><td>Protein gel electrophoresis and blotting apparatus&amp;nbsp;</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>DynaMag-2 magnet&amp;nbsp;</td><td>Life Technologies</td><td></td><td></td></tr></tbody></table>

detection of rna-binding proteins by in vitro rna pull-down in adipocyte culture

proteínas de unión a ARN (prácticas comerciales restrictivas) están emergiendo como una capa de regulación en el desarrollo y la función del tejido adiposo. Prácticas comerciales restrictivas juegan un papel clave en la regulación de la expresión génica a nivel posttranscriptional al afectar la eficiencia de la estabilidad y de la traducción de ARNm diana. técnica desplegable RNA ha sido ampliamente utilizado para estudiar la interacción de ARN-proteína, que es necesaria para dilucidar el mecanismo de la función de las prácticas comerciales restrictivas &quot;, así como largos ARN no codificante&quot; (lncRNAs) subyacente. Sin embargo, la alta abundancia de lípidos en los adipocitos plantea un desafío técnico en la realización de este experimento. Aquí un protocolo desplegable ARN detallada está optimizado para el cultivo de adipocitos primaria. Un fragmento de ARN a partir de (AR) 3 &#39;no traducida del receptor de andrógenos (3&#39;UTR) que contiene un elementwas adenilato-uridylate ricos utilizados como un ejemplo para demostrar cómo recuperar su socio RBP, proteínas Hur, de lystate adipocito. El método descrito aquí se puede aplicar para detectar las interacciones entrePrácticas comerciales restrictivas y ARN no codificantes, así como entre las prácticas comerciales restrictivas y ARN de codificación.

En este experimento de demostración, un fragmento de ARN AR se utilizó como cebo para capturar su proteína de unión Hur. Tanto un ARN cebo FL que no es relevante para la proteína Hur, y una alícuota de perlas de estreptavidina en blanco no conjugados sirvieron como controles negativos. interacciones ARN-proteína puede ocurrir ya sea en el núcleo o citoplasma, y ​​este protocolo desplegable se puede aplicar a total o fraccionado lisados ​​celulares (citoplásmicos o nuclea...

Watch this Scientific Journal Video about Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture at JoVE.com

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

Un protocolo desplegable ARN se optimiza aquí para la detección de interacciones entre proteínas de unión a ARN (prácticas comerciales restrictivas) y no codificante, así como los ARN de codificación. Un fragmento de ARN de receptor de andrógenos (AR) se utilizó como un ejemplo para demostrar cómo recuperar su RBP de lystate de adipocitos marrones primarios.

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 ...

detection-rna-binding-proteins-vitro-rna-pull-down-adipocyte

Research

JoVE Journal

Biology

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

23.5K vistas.  Duke-NUS Medical School.  Un protocolo desplegable ARN se optimiza aquí para la detección de interacciones entre proteínas de unión a ARN (prácticas comerciales restrictivas) y no codificante, así como los ARN de codificación. Un fragmento de ARN de receptor de andrógenos (AR) se utilizó como un ejemplo para demostrar cómo recuperar su RBP de lystate de adipocitos marrones primarios. 

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

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

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Preparación de muestras para identificación basada en espectrometría de masa de regiones de unión de ARN

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Bioquímica

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 ...

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA&#39;s family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co- and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That&#39;s the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest.
This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

This RNA pull-down method allows identifying the RNA targets of a long non-coding RNA (lncRNA). Based on the hybridization of home-made, designed anti-sense DNA oligonucleotide probes specific to this lncRNA in an appropriately fixed tissue or cell line, it efficiently allows the capture of all RNA targets of the lncRNA.

Procedimiento de ARN Pull-down para identificar dianas de ARN de un ARN largo no codificante

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

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

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 structures and motifs. In this RNA-pulldown protocol, commercially synthesized RNA probes are labeled with a modified form of biotin, desthiobiotin, at the 3' terminus of the RNA strand, which reversibly binds to streptavidin and thus allows elution of proteins under more physiological conditions. The RNA-desthiobiotin is immobilized through interaction with streptavidin on magnetic beads, which are used to pull down proteins that specifically interact with the RNA of interest. Non-denatured and active proteins from the cytosolic fraction of mesothelioma cells are used as the source of proteins. The method described here can be applied to detect the interaction between known RNA binding proteins and a 25-nucleotide (nt) long RNA probe containing a sequence of interest. This is useful to complete the functional characterization of stabilizing or destabilizing elements present in RNA molecules achieved using a reporter vector assay.

Desthiobiotin labeling of a synthetic 25-nucleotide RNA oligo, which contains an adenine-rich element (ARE) motif, allows specific binding of cytosolic ARE-binding protein.

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

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 ...

Investigación sobre el cáncer

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; ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

Un ensayo para cuantificar la unión de proteínas y ARN en bacterias

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.

An easy-to-use RNA pull-down protocol is designed for the identification of RNAs engaged in direct RNA/RNA interaction with a long non-coding RNA. The protocol uses psoralen as a fixative to cross-link only RNA/RNA interactions and provides the whole direct RNA interactome of a long non-coding RNA when coupled with RNA sequencing.

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome ...

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 ...

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

Un modelo de cultivo celular de adipocitos para estudiar el impacto de la modulación de proteínas y microARN en la función de los adipocitos

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

La detección de las proteínas de unión a ARN por

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