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Un nuevo espliceosoma que contiene U11, U12 y U5 snRNP extirpa un intrón de clase menor (AT-AC) in vitro
1Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
Cell
|March 8, 1996
Resumen
Los investigadores identificaron una clase menor de intrones AT-AC en los genes de metazoos. Un sistema in vitro utilizando células HeLa empalmó con precisión estos intrones, revelando las funciones esenciales de las pequeñas ribonucleoproteínas nucleares U12 y U5 (snRNP) en el espliceosoma AT-AC.
Área de la Ciencia:
- Biología Molecular Biología Molecular
- Genética La genética.
- El Splicing de ARN y ARN.
Sus antecedentes:
- Un subconjunto de intrones en los genes que codifican las proteínas de los metazoos utilizan el sitio de empalme no canónico (AT-AC) y las secuencias del sitio de ramificación.
- Estos intrones AT-AC son menos comunes que los intrones canónicos, pero juegan un papel en la expresión génica.
Objetivo del estudio:
- Establecer un sistema in vitro para el estudio del empalme intronico AT-AC.
- Para aclarar los mecanismos moleculares e identificar los componentes clave involucrados en el empalme AT-AC.
Principales métodos:
- Desarrollo de un sistema de empalme in vitro basado en células HeLa utilizando un sustrato de pre-ARNm con un intrón AT-AC del gen P120 humano.
- Confirmación de las interacciones predichas en el sitio de la rama utilizando el enlace cruzado de psoraleno.
- Análisis del ensamblaje del espliceosoma utilizando electroforesis en gel nativo.
- Evaluación funcional de los snRNP a través de la inhibición mediada por oligonucleótidos.
Principales resultados:
- El sistema in vitro establecido empalmó con precisión el intrón P120 AT-AC.
- El empalme procedió a través de un lariat intermedio, con el sitio de la rama Un residuo pronosticado para sobresalir de un dúplex con U12 pequeña ribonucleoproteína nuclear (snRNP).
- Se demostró que los snRNP U11, U12 y U5 se ensamblan en complejos de empalme en el pre-ARNm P120.
- Los estudios de inhibición confirmaron las funciones esenciales de los snRNP U12 y U5 en la función del espliceosoma AT-AC.
Conclusiones:
- El estudio caracterizó con éxito un sistema in vitro para el empalme de intrones AT-AC.
- Los snRNP U12 y U5 son componentes críticos del espliceosoma AT-AC, que participan en el empalme de intrones no canónicos.
- Este trabajo proporciona información sobre la maquinaria y los mecanismos que rigen las vías alternativas de empalme.
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