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PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins
Published on: July 2, 2010
Argonaute HITS-CLIP decodifica los mapas de interacción de microARN y ARNm de las imágenes
Sung Wook Chi1, Julie B Zang, Aldo Mele
1Laboratory of Molecular Neuro-Oncology and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA.
Nature
|June 19, 2009
Resumen
La secuenciación de alto rendimiento de ARN aislados por inmunoprecipitación de enlace cruzado (HITS-CLIP) identificó las interacciones de microARN (miRNA) y ARN mensajero objetivo (mRNA) en el cerebro de ratón. Este método traza mapas de los sitios de unión del miARN, lo que hace avanzar la comprensión de la regulación génica.
Área de la Ciencia:
- Biología Molecular Biología Molecular
- La genómica es la genómica.
- La neurociencia es la neurociencia.
Sus antecedentes:
- Los microARN (miARN) regulan la expresión génica a través del emparejamiento de bases con los ARN mensajeros (ARNm).
- Predecir las interacciones miRNA-mRNA es un desafío debido a la secuencia corta (6-8 nucleótidos) requerida para la unión.
- La secuenciación de alto rendimiento de ARN aislados por inmunoprecipitación de enlace cruzado (HITS-CLIP) identifica las interacciones funcionales proteína-ARN.
Objetivo del estudio:
- Para usar HITS-CLIP para mapear los sitios de unión de miRNA y mRNA in vivo.
- Para identificar las interacciones específicas miRNA-mRNA en el cerebro del ratón.
- Proporcionar una plataforma para explorar la acción del miRNA y dirigirse a las interacciones clínicamente relevantes.
Principales métodos:
- Enlace cruzado covalente de complejos proteico-ARN nativos de argonauta (Ago) en el cerebro de ratón.
- Utilizando HITS-CLIP para generar simultáneamente datos del sitio de unión de Ago-miRNA y Ago-mRNA.
- Análisis bioinformático para identificar los sitios de interacción miRNA-mRNA.
- Validación de mapas de interacción de todo el genoma para miR-124 y mapeo de 20 miRNAs abundantes.
Principales resultados:
- Generó datos simultáneos del sitio de unión de Ago-miRNA y Ago-mRNA del cerebro de ratón.
- Se identificaron mapas de interacción de todo el genoma para miR-124.4.
- Creación de mapas de interacción para los 20 miRNA más abundantes en el cerebro de ratón P13.
- Se establecieron secuencias precisas para la orientación miRNA-mRNA.
Conclusiones:
- Ago HITS-CLIP es una plataforma versátil para estudiar la especificidad y la función del miRNA in vivo.
- Este enfoque permite la identificación de las interacciones precisas miRNA-mRNA.
- Los hallazgos facilitan la orientación de las interacciones miRNA-mRNA clínicamente relevantes.
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