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NET-Seq de mamíferos revela transcripción naciente en todo el genoma acoplada al procesamiento de ARN

Takayuki Nojima¹, Tomás Gomes², Ana Rita Fialho Grosso²

¹Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.

|April 25, 2015

Resumen

Desarrollamos mNET-seq para visualizar la transcripción naciente en mamíferos. Esta tecnología revela nuevos detalles sobre el procesamiento del ARN, la terminación y cómo la fosforilación de la ARN polimerasa II CTD difiere entre las levaduras y los mamíferos.

Área de la Ciencia:

Biología Molecular Biología Molecular
Genética La genética.
La bioquímica es la bioquímica.

Sus antecedentes:

La transcripción es un proceso biológico complejo y dinámico.
Comprender la transcripción naciente es crucial para descifrar la regulación génica.
Las tecnologías existentes tienen limitaciones para proporcionar una resolución de un solo nucleótido de la transcripción activa.

Objetivo del estudio:

Desarrollar y validar una nueva tecnología para perfilar la transcripción naciente en células de mamíferos.
Para investigar la dinámica de la ARN polimerasa II (Pol II) durante la transcripción y el procesamiento del ARN.
Para aclarar los roles de la fosforilación de CTD y los factores de terminación en la transcripción de mamíferos.

Principales métodos:

Desarrollo de la tecnología de secuenciación de transcripción de alargamiento nativo para la cromatina de mamíferos (mNET-seq).
Perfilado de resolución de un solo nucleótido del ARN naciente y el Pol II asociado.
Análisis de los intermediarios de procesamiento de ARN, incluida la actividad del espliceosoma.
Investigación del impacto del agotamiento del factor de terminación en la pausa y terminación de Pol II.

Principales resultados:

mNET-seq proporciona instantáneas dinámicas de alta resolución de la transcripción naciente.
Se detectó escisión en el sitio de 5'splice y asociación con Pol II CTD fosforilado en S5P.
Se demostró que los factores de terminación influyen en la pausa de Pol II y restringen la transcripción no productiva.
Se revelaron diferencias significativas en los patrones de fosforilación de CTD entre la transcripción de levaduras y mamíferos.

Conclusiones:

mNET-seq es una poderosa herramienta para estudiar la dinámica de la transcripción de los mamíferos.
Los hallazgos proporcionan nuevos conocimientos sobre la interacción entre la transcripción, el procesamiento de ARN y la terminación.
La transcripción de los mamíferos exhibe mecanismos reguladores distintos en comparación con la levadura, particularmente en lo que respecta a la fosforilación de la CTD.

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