Agradecemos a los miembros del laboratorio Tuschl útil para los debates. MH es apoyada por la Deutscher Akademischer Austauschdienst (DAAD). Este trabajo fue apoyado por el Fondo Nacional de la Subvención Suiza # 3100A0-114001 a MZ, TT es un investigador del HHMI, y trabajar en su laboratorio fue apoyado por subvenciones del NIH GM073047 y MH08442 y la Fundación Starr.

El protocolo se describe el procedimiento PAR-clip para HEK293 células que expresan FLAG / HA-etiquetados IGF2BP1 a la inducción con doxiciclina. Vamos a utilizar un anticuerpo anti-FLAG de inmunoprecipitación. PAR-clip funciona con cualquier línea celular que expresa niveles detectables de la endógena, sin etiquetar las proteínas de unión a ARN (RBP) de interés si un anticuerpo eficaz para la inmunoprecipitación está disponible. Las células en expansión <ol><li> Ampliar FlpIn-HEK293/TO/FLAG/HA-IGF2BP1 células en un medio de crecimiento. Se recomienda utilizar entre 100 a 400 x 10 6 células (aprox. 10 a 40 15 cm placas de cultivo celular) como punto de partida. Creciendo a una confluencia de aproximadamente el 80%. </li><li> 14 h antes de añadir un entrecruzamiento) 4-thiouridine a una concentración final de 100 mM (1:1000 v / v de M 1 4-thiouridine solución madre) directamente en el medio de cultivo celular y b) inducir la expresión de la Bandera / HA etiquetados IGF2BP1 mediante la adición de 1 mg / ml de doxiciclina (1:10.000 v / v de 10 mg / ml de solución de doxiciclina). NOTA: en lugar de 4-tiouridina también se puede utilizar 100 mM de 6-thioguanosine. </li></ol> UV-entrecruzamiento <ol><li> Lave las células una vez con 10 ml de helado de PBS por placa y eliminar por completo PBS. </li><li> Coloque las placas en una bandeja con hielo y descubierto irradian con luz nm 0,15 J / cm 2 de 365 UV en un Stratalinker 2400 (Stratagene) o dispositivo similar. </li><li> Raspar las células con una goma de policía en 1 ml de PBS por placa, la transferencia de tubos de 50 ml centrifugación y recoger por centrifugación a 500 xg durante 5 min a 4 ° C y descartar el sobrenadante. 100 x 10 6 células HEK293 (10 15 placas cm) rendirá aproximadamente. 1 ml de celda húmeda de pellets. </li><li> (Opcional) A menos que usted desea continuar directamente con la lisis celular, descarga congelar el sedimento de células en nitrógeno líquido y se almacenan a -80 ° C. Gránulos de las células se pueden almacenar durante al menos 12 meses. </li></ol> Lisis celular y digerir RNaseT1 <ol><li> Toma sedimento celular de las células reticuladas en 3 volúmenes de tampón de lisis 1x NP40 e incubar en hielo durante 10 minutos. </li><li> Lisado de células claras por centrifugación a 13.000 xg durante 15 min a 4 ° C. </li><li> Borrar el lisado más filtrándola a través de un filtro de 0,2 micras jeringa membrana (Acrodisc de Pall o equivalente). </li><li> Añadir RNasa T1 (Fermentas, 10.000 U / l) a una concentración final de 1 U / l, y se incuban en un baño de agua durante 15 minutos a 22 ° C. Fría reacción después de 5 min en hielo antes de proceder. </li></ol> Inmunoprecipitación y la recuperación de reticulado objetivo fragmentos de ARN Usando el separador magnético Siga estas pautas a través de la preparación de la muestra para evitar que las cuentas magnéticas que se sequen. <ol><li> Coloque el tubo que contiene las cuentas en el soporte magnético de 1 2 minutos. </li><li> Añadir la solución tampón en el tubo, mientras que el tubo está en el separador magnético. </li><li> Tapar el tubo, retírelo del separador magnético, y volver a suspender las bolas. Puede volver a suspender las cuentas de prender el tubo con el dedo o el uso de un vórtice del 5 6. </li><li> Centrifugar brevemente para recoger los granos que pueden permanecer en la tapa del tubo. </li><li> Repita los pasos 1 a 4 según sea necesario. </li></ol> Preparación de partículas magnéticas <ol><li> Transferencia de 10 l de Dynabeads proteína G partículas magnéticas (Invitrogen) por lisado celular ml (para un experimento típico debe ser de aprox. 40 50 l de bolas) a un tubo de microcentrífuga de 1,5 ml. Lávese las cuentas dos veces con 1 ml de solución tampón de citrato-fosfato. </li><li> Resuspender en el doble del volumen de tampón de citrato-fosfato en relación con el volumen original de la suspensión de cuentas. </li><li> Añadir 0,25 g de anti-FLAG anticuerpo monoclonal M2 (Sigma) por cada ml de suspensión y se incuban en una rueda giratoria de 40 min a temperatura ambiente. </li><li> Lávese las cuentas dos veces en 1 ml de solución tampón de citrato-fosfato para eliminar el anticuerpo no unido. </li><li> Volver a suspender las cuentas en el doble del volumen de tampón de citrato-fosfato en relación con el volumen original de la suspensión de perlas. </li></ol> Inmunoprecipitación (IP), en segundo lugar la digestión RNasa T1, y desfosforilación <ol><li> Añadir 20 l de recién preparada anticuerpo conjugado con partículas magnéticas, por ml de lisado parcial RNasa T1 de células tratadas y se incuba en tubos de 15 ml centrifugación en una rueda giratoria de 1 hora a 4 ° C. </li><li> Recoger partículas magnéticas en un colector de partículas magnéticas de 15 y 50 tubos de centrifugación ml (Invitrogen) y la transferencia a tubos de 1,5 ml de microfuga. </li><li> Lávese las cuentas 3 veces en 1 ml de solución de lavado IP. </li><li> Añadir RNaseT1 (Fermentas, 10.000 U / l) a una concentración final de 100 U / l, y se incuba la suspensión de bolas en un baño de agua durante 15 minutos a 22 º C. Cool posteriormente en el hielodurante 5 min. </li><li> Lávese las cuentas 3 veces en 1 ml de solución de lavado de alta en sal. </li><li> Cuentas de volver a suspender en un volumen de tampón desfosforilación </li><li> Añadir la pantorrilla intestinal fosfatasa alcalina (CIAP, NEB) a una concentración final de 0,5 U / l, y se incuba la suspensión durante 10 minutos a 37 ° C. </li><li> Lávese las cuentas dos veces en 1 ml de solución de lavado fosfatasa </li><li> Lávese las cuentas dos veces en la polinucleótido quinasa (PNK) de amortiguación sin TDT (la concentración de la TDT es necesario para la reacción enzimática es lo suficientemente alta como para dañar los granos magnéticos). </li><li> Volver a suspender las cuentas de un volumen original de cuentas de PNK buffer </li></ol> Radiomarcado de segmentos de ARN a las proteínas reticuladas immunoprecipitated <ol><li> A la suspensión de bolas se ha descrito anteriormente, añadir Υ-32 P-ATP a una concentración final de 0,5 Ci / mL y T4 PNK (NEB) y 1 U / l en un volumen de grano original. Incubar la suspensión durante 30 minutos a 37 ° C. </li><li> Añadir ATP no radiactivo para obtener una concentración final de 100 mM e incubar durante otros 5 minutos a 37 ° C. </li><li> Lavado de los granos magnéticos 5 veces con 800 l de buffer PNK sin TDT. </li><li> Volver a suspender las bolas en 70 l de tampón de carga SDS-PAGE. </li></ol> SDS-PAGE y electroelución de reticulado ARN-proteína de rodajas de gel <ol><li> Incubar la suspensión radiomarcado durante 5 minutos en un bloque de calor a 95 ° C para desnaturalizar las prácticas comerciales restrictivas y la liberación de la immunoprecipitated con ARN reticulado y agitar. </li><li> Eliminar las cuentas en el separador magnético y la transferencia del sobrenadante a un tubo de microcentrífuga limpio de 1.5 ml. </li><li> Carga 40 ml del sobrenadante por pocillo de una Novex Bis-Tris 4.12% (Invitrogen) en gel de poliacrilamida prefabricados y correr el gel durante 55 min a 200 V. </li><li> Desmonte la cámara de gel y desmantelar el gel, dejándola montado en una placa. Para facilitar la alineación del gel de la hoja impresa PhosphorImager, se recomienda la implantación de tres pequeñas piezas de gel radiactivos de forma asimétrica en tres de las cuatro esquinas del gel. Piezas radiactivas gel se pueden recoger a partir de geles que se utilizaban anteriormente para purificar radiomarcado oligonucleótidos sintéticos. Envuelva el gel en la película de plástico (por ejemplo, Saran wrap) para evitar la contaminación. </li><li> Exponer el gel a una pantalla PhosphorImager blanqueó durante 1 hora y visualizar en un phosphorimager. </li><li> Alinear el gel en la parte superior de la impresión PhosphorImager con las piezas de gel implantado para la orientación. Cortar las bandas que se corresponden con el tamaño esperado de las prácticas comerciales restrictivas (IGF2BP1, aprox. 75 kDa) y transferir a un tubo de D-Tubo dializador Midi y añadir 800 l 1x buffer SDS funcionamiento. </li><li> Electroelute el reticulado de ARN-RBP complejo en 1x SDS funcionamiento de amortiguación de 100 V durante 2 h. extirpados de la gelatina y se electroeluye en Midi D-Tubo dializador (Novagen) en 800 l de amortiguación SDS funcionamiento de acuerdo a las instrucciones del fabricante. </li></ol> Proteinasa K digestión <ol><li> Añadir un volumen igual de tampón de proteinasa K 2 veces con respecto a la electroeluate, seguido por la adición de proteinasa K (Roche) a una concentración final de 1,2 mg / ml. Incubar durante 30 min a 55 ° C. </li><li> Recuperar el ARN de ácido fenol / cloroformo / extracción de la IAA (25:24:1, pH 4,0), seguido de una extracción con cloroformo. Añadir 1 l de glucógeno (10 mg / ml) precipitar el ARN mediante la adición de 3 volúmenes de etanol. Disolver el precipitado en 10,5 l de agua. </li></ol> preparación de la biblioteca de cDNA y la secuencia de profundidad Llevar el ARN recuperado a través de un protocolo estándar de preparación de la biblioteca de cDNA descrito originalmente para la clonación de pequeños ARNs reguladores 19. El primer paso, la ligadura de 3 &#39;adaptador, se llevó a cabo como se describe en una escala de 20 l con 10,5 l de la ARN recuperado. Utilice el adaptador de juegos Solexa secuenciación descritos. Dependiendo de la cantidad de ARN se recuperó, 5&#39;-adaptador-3&#39;-adaptador de los productos sin insertos pueden ser detectados después de la amplificación del cDNA adicionales como las bandas de PCR. En tales casos, el consumo del producto ya PCR del tamaño esperado de un 3% NuSieve bajo punto de fusión en gel de agarosa, eluir el producto de PCR de las piezas de gel con el kit de GelElute (Qiagen) y la secuencia de uso de la tecnología Solexa. Una secuencia Solexa ejecutar normalmente ofrece entre 6 y 10 millones de secuencia se lee que son suficientes para una cobertura amplia del transcriptoma de los sitios de unión de las proteínas de unión a ARN. Bioinformáticas de análisis Un cuidadoso análisis bioinformático de la secuencia se lee que hay que hacer para obtener puntos de vista significativos en los sitios de unión a ARN para el RBP examinado, como el elemento de reconocimiento de ARN, las regiones preferidas de unión de la RBP ha (exonic vs intrónicas, la secuencia de codificación frente a traducir secuencia). La secuencia se lee necesitan estar alineados en contra de las bases de datos del genoma y EST. Por lo general, el uso lee Mapping única en el genoma con un máximo de un desajuste, inserción o deleción de construir grupos de la secuencia se lee que luego pueden ser analizados. La frecuencia de mutaciones características de la secuencia se lee en clúster, T a las transiciones cuando se utiliza C 4-SU y G para una transición al uso de 6-SG, son indicativos de las secuencias de éxito reticulado. En nuestra experiencia ARN reticulado etiquetados con 4-SU muestran una tasa de mutación de fondo de aproximadamente un 20%. Esta tasa se incrementa hasta aprox. 50-80% en la reticulación. Una descripción detallada de los análisis bioinformático se puede encontrar en el material complementario de la publicación por Hafner et al. 18 Pasos opcionales Determinación de los niveles de incorporación de 4-tiouridina en el ARN total Aislar el ARN total de la línea celular estable que expresa la RBP de interés después de haber crecido en medio suplementado con 100 mM 4SU 16 h antes de la cosecha. Como control, las células de la cosecha crecido sin adición 4SU. Aislar el ARN total mediante la adición de 3 volúmenes de reactivo Trizol (Sigma) a los gránulos de las células lavadas siguiendo las instrucciones del fabricante s. Además era purificar el ARN total utilizando Qiagen RNeasy de acuerdo con el protocolo del fabricante. Para evitar la oxidación de 4SU durante el aislamiento de ARN y análisis, añadir 0,1 mM ditiotreitol (TDT) a los búferes de lavado y posterior pasos enzimáticos. Digerir y desfosforilado ARN total a los nucleósidos única para el análisis de HPLC como se describe antes de los 20. En pocas palabras, en un volumen de 30 l, incubar 40 microgramos de RNA total se purifica por 16 horas a 37 ° C con 0,4 U de fosfatasa alcalina bacteriana (Bioquímica Worthington) y 0,09 U veneno de la serpiente de la fosfodiesterasa (Worthington Biochemical). Como patrón de referencia, utilizar un ARN sintético 4SU marcado, (que de forma estándar el uso CGUACGCGGAAUACUUCGA (4SU) U) y también está sujeto a completar la digestión enzimática. Separar las mezclas resultantes de ribonucleósidos por HPLC en un descubrimiento Supelco C18 (partículas de sílice de fase en régimen de servidumbre M 5, 250 x 4,6 mm) de columna de fase inversa (Bellefonte PA, EE.UU.). Buffers HPLC son de 0,1 M TEAA en el 3% de acetonitrilo (A) y el 90% de acetonitrilo en agua (B). Utilice un gradiente isocrático: 0% de B durante 15 min, 0 a 10% de B durante 20 min, de 10 a 100% B durante 30 min. Aplicar un 5 min 100% B de lavado aplicado entre las corridas de limpiar la columna HPLC. Resultados representante <img src="/files/ftp_upload/2034/2034fig1.jpg" alt="Figura 1" /> Figura 1 (panel derecho) muestra un resultado representativo de un PAR-clip realizado con líneas celulares que expresan FLAG / HA-etiquetados IGF2BP1 con 4-SU y 6 SG-. Tenga en cuenta que la eficiencia de reticulación de 6-SG para IGF2BP1 es menor que la eficiencia de entrecruzamiento de 4-SU. La eficiencia de entrecruzamiento menor resultará en un fondo más alto de las secuencias derivadas de fragmentos de ARN celular abundante y por lo tanto debe tener en cuenta la ampliación de la experiencia al usar nucleósidos fotorreactivos menos eficiente. El panel izquierdo de la figura 1 muestra una comparación del uso de diferentes análogos de uridina fotorreactivos que podrían ser potencialmente utilizados para PAR-clip en comparación con el tradicional reticulación UV de 254 nm. La intensidad de la banda radiactiva de la longitud correcta le da una buena idea si el experimento PAR-Clip ha trabajado y se han aislado de ARN suficiente para llevar a cabo un pequeño protocolo de la secuencia de ARN (paso a paso la descripción de la preparación de la pequeña biblioteca de cDNA secuenciación de ARN se pueden encontrar en 19). La frecuencia de mutaciones características de la secuencia se lee, T a las transiciones cuando se utiliza C 4-SU y G para una transición al uso de 6-SG, son indicativos de las secuencias de éxito reticulado. En nuestra experiencia ARN reticulado etiquetados con 4-SU muestran una tasa de mutación de fondo de aproximadamente un 20%. Esta tasa se incrementa hasta aprox. 50-80% en la reticulación.

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Base+composition+analysis+of+nucleosides+using+HPLC.">Base composition analysis of nucleosides using HPLC.</a> Current Protocols in Nucleic Acid Chemistry. Chapter 10 (Unit 10.6), (2001).</li></ol>

TT es un asesor cofundador y científico de Alnylam Pharmaceuticals y asesor de Regulus Therapeutics.

Tampones y reactivos Crecimiento a medio HEK293 <ul><li> DMEM </li><li> 10% de SFB </li><li> 2 mM L-Glutamina </li><li> 100 U / ml de penicilina </li><li> 100 U / ml de estreptomicina </li><li> 100 mg / ml de higromicina </li><li> 15 mg / ml blasticidin </li></ul> 4-tiouridina solución madre (1 M) <ul><li> 260,27 mg de 4 thiouridine </li><li> 1 ml de DMSO </li></ul> Doxiciclina acciones (10 mg / ml) <ul><li> 10 mg doxiciclina </li><li> 1 ml de DMSO </li></ul> 1x tampón de lisis NP40 Prepare un inventario de buffer 5x sin TDT y los inhibidores de la proteasa. Añadir la TDT y inhibidor de la proteasa directamente antes del experimento. <ul><li> HEPES 50 mM, pH 7,5 </li><li> 150 mM KCl </li><li> 2 mM EDTA </li><li> 1 mM NaF </li><li> 0,5% (v / v) NP40 </li><li> 0,5 mM DTT </li><li> completa EDTA libre de cóctel inhibidor de la proteasa (Roche) </li></ul> Tampón citrato-fosfato <ul><li> 4,7 g / l de ácido cítrico </li><li> 9,2 g / l de Na 2 HPO 4 </li><li> pH 5,0 </li></ul> IP-tampón de lavado <ul><li> 50 mM HEPES-KOH, pH 7,5 </li><li> 300 mM KCl </li><li> 0,05% (v / v) NP40 </li><li> 0,5 mM DTT (añadir directamente antes del experimento) </li><li> completa EDTA libre de cóctel inhibidor de la proteasa (Roche) (añadir directamente antes del experimento) </li></ul> Alto contenido de sal tampón de lavado <ul><li> 50 mM HEPES-KOH, pH 7,5 </li><li> 500 mM KCl </li><li> 0,05% (v / v) NP40 </li><li> 0,5 mM DTT (añadir directamente antes del experimento) </li><li> completa EDTA libre de cóctel inhibidor de la proteasa (Roche) (añadir directamente antes del experimento) </li></ul> Desfosforilación buffer <ul><li> 50 mM Tris-HCl, pH 7,9 </li><li> 100 mM NaCl </li><li> 10 mM de MgCl 2 </li><li> 1 mM DTT </li></ul> Fosfatasa tampón de lavado <ul><li> 50 mM Tris-HCl, pH 7,5 </li><li> 20 mM EGTA </li><li> 0,5% (v / v) NP40 </li></ul> Polinucleótido quinasa (PNK) de amortiguación sin TDT <ul><li> 50 mM Tris-HCl, pH 7,5 </li><li> 50 mM NaCl </li><li> 10 mM de MgCl 2 </li></ul> PNK buffer <ul><li> 50 mM Tris-HCl, pH 7,5 </li><li> 50 mM NaCl </li><li> 10 mM de MgCl 2 </li><li> 5 mM DTT </li></ul> SDS-PAGE de tampón de carga <ul><li> 10% de glicerol (v / v) </li><li> 50 mM Tris-HCl, pH 6,8 </li><li> 2 mM EDTA </li><li> SDS al 2% (w / v) </li><li> 100 mM DTT </li><li> 0,1% azul de bromofenol </li></ul> 2x proteinasa K buffer <ul><li> 100 mM Tris-HCl, pH 7,5 </li><li> 150 mM NaCl </li><li> 12,5 mM EDTA </li><li> 2% (w / v) SDS </li></ul>

par-clip - a method to identify transcriptome-wide the binding sites of rna binding proteins

Transcripciones de ARN que son objeto de regulación post-transcripcional de genes interactuando con cientos de proteínas de unión a ARN (las prácticas comerciales restrictivas) y microRNA que contienen complejos de ribonucleoproteína (miRNPs) que se expresan a menudo en una celda de tipo dependiente. Para entender cómo la interacción de estos factores de unión al ARN afecta a la regulación de las transcripciones individuales, mapas de alta resolución en vivo de las interacciones proteína-ARN son necesarios 1. Una combinación de métodos genéticos, bioquímicos y de cálculo se aplican normalmente para identificar las prácticas comerciales restrictivas o ARN interacciones RNA-RNP. Microarrays de perfiles de ARN asociada a las prácticas comerciales restrictivas inmunopurificados (RIP-Chip) 2 define los objetivos a nivel del transcriptoma, pero su aplicación se limita a la caracterización de las interacciones cinéticamente estable y sólo en casos raros 3,4 permite identificar el elemento de reconocimiento de las prácticas comerciales restrictivas (RRE ) dentro de la meta de ARN de largo. Objetivo más directo RBP información del sitio se obtiene mediante la combinación de reticulación UV vivo con 5,6 inmunoprecipitación 7-9 seguido por el aislamiento de reticulado segmentos de ARN y secuenciación de cDNA (CLIP) 10. El vídeo fue utilizado para identificar los objetivos de una serie de prácticas comerciales restrictivas 11-17. Sin embargo, CLIP está limitada por la baja eficiencia de la radiación UV 254 nm ARN-proteína entrecruzamiento, y la ubicación de la reticulación no es fácilmente identificable dentro de los fragmentos secuenciados reticulado, por lo que es difícil separar objetivo UV reticulado segmentos de ARN de fondo no reticulado fragmentos de ARN también está presente en la muestra. Hemos desarrollado un poderoso celular enfoque basado en los enlaces cruzados para determinar con alta resolución y transcriptoma en todo los sitios de unión de las prácticas comerciales restrictivas celular y miRNPs que llamamos PAR-Clip (fotoactivables-ribonucleósido-Enhanced entrecruzamiento e inmunoprecipitación) (ver fig. 1A de un esquema del método). El método se basa en la incorporación de los análogos de ribonucleósido fotorreactivos, tal como 4-thiouridine (4-SU) y 6-thioguanosine (6-SG) en el naciente transcripciones de ARN de las células vivas. La irradiación de las células por la luz UV de 365 nm induce reticulación eficiente de fotorreactivos etiquetados nucleósidos ARN celular a las prácticas comerciales restrictivas que interactúan. Inmunoprecipitación de las prácticas comerciales restrictivas de interés es seguido por el aislamiento del ARN reticulado y coinmunoprecipitó. El ARN aislado se convierte en una biblioteca de cDNA y la secuencia de profundidad utilizando la tecnología Solexa. Un rasgo característico de las bibliotecas de cDNA preparado por PAR-Clip es que la posición precisa de entrecruzamiento puede ser identificado por las mutaciones que residían en la secuencia de cDNA. Cuando se utiliza 4-SU, secuencias reticulado timidina a la transición citidina, mientras que con 6-SG resultados de guanosina a la adenosina mutaciones. La presencia de las mutaciones en las secuencias de reticulado permite separarlos de los antecedentes de las secuencias derivadas de abundantes ARN celular. La aplicación del método a una serie de diversas proteínas de unión a ARN se informó en Hafner et al. 18

Watch this Scientific Journal Video about PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins at JoVE.com

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

Transcripciones de ARN están sujetas a numerosas normas posttranscriptional que está mediada por una multitud de trans-acción ARN-proteínas de unión (prácticas comerciales restrictivas). Aquí presentamos un método generalizable para identificar con precisión y en una escala de transcriptoma en todo los sitios de unión a ARN de las prácticas comerciales restrictivas.

PAR-Clip - un método para identificar transcriptoma en todo los sitios de unión de las proteínas de unión a ARN

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

par-clip-method-to-identify-transcriptome-wide-binding-sites-rna

Research

JoVE Journal

Biology

53.2K vistas.  Rockefeller University. Transcripciones de ARN están sujetas a numerosas normas posttranscriptional que está mediada por una multitud de trans-acción ARN-proteínas de unión (prácticas comerciales restrictivas). Aquí presentamos un método generalizable para identificar con precisión y en una escala de transcriptoma en todo los sitios de unión a ARN de las prácticas comerciales restrictivas.

Video: PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Actina Co-sedimentación de ensayo, pues el análisis de proteína de unión a la F-actina

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Investigación

Biología

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Proteómica para identificar proteínas que interactúan con los canales de cationes P2X2 ligando

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Perfiles de metiltransferasas y Otros S-Adenosil- L-Homocisteína proteínas que se unen mediante espectrometría de masas de captura compuesto (MCP)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host macromolecule. ITC has some advantages over common spectroscopic approaches for studying host/ligand interactions. For example, the heat released or absorbed when the two components interact is directly measured and does not require any exogenous reporters. Thus the binding enthalpy and the association constant (Ka) are directly obtained from ITC data, and can be used to compute the entropic contribution. Moreover, the shape of the isotherm is dependent on the c-value and the mechanistic model involved. The c-value is defined as c = n[P]tKa, where [P]t is the protein concentration, and n is the number of ligand binding sites within the host. In many cases, multiple binding sites for a given ligand are non-equivalent and ITC allows the characterization of the thermodynamic binding parameters for each individual binding site. This however requires that the correct binding model be used. This choice can be problematic if different models can fit the same experimental data. We have previously shown that this problem can be circumvented by performing experiments at several c-values. The multiple isotherms obtained at different c-values are fit simultaneously to separate models. The correct model is next identified based on the goodness of fit across the entire variable-c dataset. This process is applied here to the aminoglycoside resistance-causing enzyme aminoglycoside N-6'-acetyltransferase-Ii (AAC(6')-Ii). Although our methodology is applicable to any system, the necessity of this strategy is better demonstrated with a macromolecule-ligand system showing allostery or cooperativity, and when different binding models provide essentially identical fits to the same data. To our knowledge, there are no such systems commercially available. AAC(6')-Ii, is a homo-dimer containing two active sites, showing cooperativity between the two subunits. However ITC data obtained at a single c-value can be fit equally well to at least two different models a two-sets-of-sites independent model and a two-site sequential (cooperative) model. Through varying the c-value as explained above, it was established that the correct binding model for AAC(6')-Ii is a two-site sequential binding model. Herein, we describe the steps that must be taken when performing ITC experiments in order to obtain datasets suitable for variable-c analyses.

ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.

Recogida de concentración variable conjuntos de datos calorimetría de titulación isotérmica para determinar los mecanismos de enlace

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

iClip - transcriptoma en todo el mapeo de la proteína-ARN interacciones con la Resolución de nucleótidos individuales

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

Pull-down of Calmodulin-binding Proteins

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca2+ (Ca2+-CaM) as opposed to its Ca2+-free state (ApoCaM)3.
While most target proteins bind Ca2+-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin4, neurogranin (Ng)5, and certain myosins6. These proteins have been shown to play important roles in presynaptic function7, postsynaptic function8, and muscle contraction9, respectively. Their ability to bind and release CaM in the absence or presence of Ca2+ is pivotal in their function. In contrast, many proteins only bind Ca2+-CaM and require this binding for their activation. Examples include myosin light chain kinase10, Ca2+/CaM-dependent kinases (CaMKs)11 and phosphatases (e.g. calcineurin)12, and spectrin kinase13, which have a variety of direct and downstream effects14.
The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca2+-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca2+-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.
Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca2+, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca2+ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca2+/CaM signaling and proteins that interact with CaM.

Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function.

Desplegable de proteínas de unión a calmodulina

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Modificado levadura sistema híbrido de dos para identificar proteínas que interactúan con el factor de crecimiento progranulina

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

El aislamiento de los ribosomas polipéptidos nacientes Bound<em&gt; In vitro</em&gt; Para la identificación de sitios de traslación pausa a lo largo del ARNm

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Purificación de proteínas libre de Método de Determinación afinidad de unión por termoforesis Microscale

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

Un método de cuantificación sensible y específica para la determinación de suero miosina cardíaca proteína de unión-C por inmunoensayo electroquimioluminiscencia

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...