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Reconocimiento funcional del sitio de empalme AG de 3' por el factor de empalme U2AF3535
1Howard Hughes Medical Institute, Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester 01605, USA.
Nature
|January 5, 2000
Resumen
La proteína del factor auxiliar U2 35 (U2AF35) se une directamente al sitio de empalme 3, un paso crucial para el ensamblaje del espliceosoma en los metazoos. Esta interacción es vital para la eficiencia de empalme, especialmente en intrones con tramos débiles de polipirimidina.
Área de la Ciencia:
- Biología Molecular Biología Molecular
- El Splicing de ARN y ARN.
- Expresión génica de la expresión génica.
Sus antecedentes:
- El ensamblaje del espliceosoma se inicia con U1 snRNP reconociendo el sitio de empalme de 5' y el factor auxiliar U2 (U2AF) que se une al tracto polipirimidino.
- U2AF es un heterodimero de U2AF65 y U2AF35; U2AF65 se une al tracto polipirimidino, pero el papel de U2AF35 en el empalme no ha sido claro a pesar de su esencialidad.
- Si bien se sabe que U2AF65 se une al tracto polipirimidino, el factor de reconocimiento inicial para el sitio de empalme 3' durante el ensamblaje del espliceosoma siguió siendo elusivo.
Objetivo del estudio:
- Para aclarar el papel de U2AF35 en el ensamblaje del espliceosoma.
- Para identificar el factor de proteína responsable del reconocimiento inicial del sitio de empalme de 3'.
- Explicar el requisito del dinucleótido AG en el sitio de empalme 3' para ciertos intrones.
Principales métodos:
- Enlace cruzado específico del sitio para detectar las primeras interacciones proteína-ARN durante el ensamblaje del espliceosoma.
- Análisis mutacional para sondear la función U2AF35.
- Selección genética in vitro para identificar las actividades de unión al ARN de U2AF35.
Principales resultados:
- U2AF35 entra directamente en contacto con el sitio de empalme 3' al principio del ensamblaje del espliceosoma.
- U2AF35 exhibe actividad de unión de ARN específica de la secuencia, reconociendo el consenso AG/G en el sitio de empalme 3'.
- La interacción del sitio de empalme U2AF35-3' es crítica para la unión y empalme de U2AF cuando los tractos de polipirimidina son débiles.
Conclusiones:
- U2AF35 posee una nueva actividad bioquímica, que se une directamente al sitio de empalme de 3'.
- U2AF35 es el factor primario que reconoce el sitio de empalme 3' durante el ensamblaje temprano del spliceosome.
- Esta interacción aclara la necesidad del dinucleótido AG para el empalme en contextos específicos de intrones.
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