Interacción de las proteínas de procesamiento con los precursores de miARN y su mecanismo en plantas
Archivos
Fecha
2015-12-14
Autores
Título de la revista
ISSN de la revista
Título del volumen
Editor
Resumen
DCL1 es la enzima ribonucleasa central en la biogénesis de micro-ARNs en plantas. Su función consiste en realizar dos cortes sucesivos sobre transcriptos primarios para liberar el micro-ARN maduro. El mecanismo a través del cual la enzima reconoce los sitios de corte sobre los precursores resta ser elucidado. La enzima presenta dos dominios de unión a ARN doble hebra en tándem en el extremo carboxilo-terminal, los cuales son esenciales para su funcionamiento in vivo.
En este trabajo de tesis nos dedicamos principalmente a la descripción biofísica del primero de los dominios de unión a ARN doble hebra de DCL1 de Arabidopsis thaliana, el cual se encuentra altamente conservado entre las distintas especies de plantas. Demostramos que el dominio libre en solución de encuentra desestructurado tanto en forma aislada como en presencia de regiones vecinas de la proteína. A pesar de estar desestructurado, es capaz de unir ARN sustrato. Mediante el análisis estructural del complejo con el ARN encontramos que el dominio adquiere una conformación plegada al entrar en contacto con el sustrato. La topología de la conformación plegada adquirida, que calculamos en base a datos de RMN, se corresponde con la esperada para un dominio de la familia de los dominios de unión a ARN doble hebra. Una vez definidas las características de este sistema procedimos a realizar, por un lado, una descripción más profunda de la forma libre desestructurada y, por otro, proponer un mecanismo posible para la unión al sustrato y plegamiento de la proteína.
De la forma libre podemos decir que globalmente es una especie flexible, aunque no se encuentra completamente desestructurada. A través de distintos observables de RMN logramos determinar que la región correspondiente al extremo carboxilo-terminal presenta una cierta rigidez y explora conformaciones tipo hélice alfa. La presencia de esta estructura preformada podría resultar esencial para la unión al sustrato y el plegamiento.
En cuanto al mecanismo de unión y plegamiento pudimos obtener evidencia que sugiere la existencia de un complejo intermediario entre la forma libre desplegada y la forma plegada unida al ARN. En este complejo intermediario la proteína se encuentra en una tercer especie, también mayormente desplegada y unida al sustrato.
Finalmente en una tercera etapa del trabajo nos abocamos al estudio desde el punto de vista estructural de precursores completos de micro-ARNs. Buscamos validar los determinantes que permiten reconocer la posición del micro-ARN dentro del precursor por parte del complejo de procesamiento formado por DCL1 y las proteínas accesorias HYL1 y SERRATE. Estudiamos cuatro precursores de diferentes familias por técnicas bioquímicas de sondeo de estructura secundaria y flexibilidad de la cadena. Logramos validar experimentalmente determinantes estructurales propuestos a partir de estructuras calculadas in silico. También encontramos indicios que sugieren que la posición del sitio del primer corte, el cual es fundamental para definir el registro de los cortes sucesivos ejecutados por DCL1, podría estar relacionada con un cambio abrupto desde una región de la hebra con una conformación flexible a una de menor flexibilidad. Esta particularidad podría estar permitiendo una interacción específica con las proteínas que conforman el complejo de procesamiento.
DCL1 is the main ribonuclease in micro-RNA biogenesis in plants. Its function consists in performing two successive cuts on primary transcripts in order to release the mature micro-RNA. The mechanism through which the enzyme recognizes the cleavage sites remains to be elucidated. The enzyme presents two double stranded RNA binding domains in tandem on the carboxylic-terminal end, which are essential for its in vivo function. In this work, we focused mainly on the biophysical description of the first double stranded RNA binding domain of DCL1 of Arabidopsis thaliana, which is highly conserved among plant species. We have demonstrated that the free domain in solution is unstructured, not only when isolated but also when surrounding regions of the protein are present. Even though it is unstructured, the domain is capable of binding to the substrate. Through the structural analysis of the complex with the RNA we found the domain acquires a folded conformation when it binds the substrate. The topology of the acquired folded conformation, calculated based on NMR data, corresponds to the double stranded RNA binding domain family. Once we defined these characteristics on the system we proceeded to perform a deeper characterization on the free unstructured form and to propose a possible mechanism for substrate binding and folding of the protein. On the free form we found that it is globally a flexible species, even though it is not completely unstructured. Through different NMR observables we have determined that the carboxylic-terminal end presents a certain rigidity and explores alfa helix conformations. This prefolded structure could be essential for binding to the substrate and folding. On the binding and folding mechanism we could obtain evidence that suggests the existence of an intermediate complex between the free unfolded form and the RNA bound folded form. In this intermediate complex the protein is also mainly unfolded but bound to the substrate. Finally, on a third stage of this work we performed a structural study of full-length micro-RNA precursors. We have focused on validating the determinants that allow the recognition of the micro-RNA position in the precursor by the processing complex involving DCL1 and the accessory proteins HYL1 and SERRATE. Employing biochemical techniques for probing secondary structure and strand flexibility we have studied four precursors from different families. We were able to experimentally validate structural determinants proposed on the basis of in silico calculated structures. We found evidence that suggests the position of the first cleavage site, which is essential for defining the register for the successive DCL1 cuts, could be related with an abrupt change from a flexible region of the strand to a much lesser flexible one. This peculiarity could be allowing for a specific interaction with the proteins involved in the processing complex.
DCL1 is the main ribonuclease in micro-RNA biogenesis in plants. Its function consists in performing two successive cuts on primary transcripts in order to release the mature micro-RNA. The mechanism through which the enzyme recognizes the cleavage sites remains to be elucidated. The enzyme presents two double stranded RNA binding domains in tandem on the carboxylic-terminal end, which are essential for its in vivo function. In this work, we focused mainly on the biophysical description of the first double stranded RNA binding domain of DCL1 of Arabidopsis thaliana, which is highly conserved among plant species. We have demonstrated that the free domain in solution is unstructured, not only when isolated but also when surrounding regions of the protein are present. Even though it is unstructured, the domain is capable of binding to the substrate. Through the structural analysis of the complex with the RNA we found the domain acquires a folded conformation when it binds the substrate. The topology of the acquired folded conformation, calculated based on NMR data, corresponds to the double stranded RNA binding domain family. Once we defined these characteristics on the system we proceeded to perform a deeper characterization on the free unstructured form and to propose a possible mechanism for substrate binding and folding of the protein. On the free form we found that it is globally a flexible species, even though it is not completely unstructured. Through different NMR observables we have determined that the carboxylic-terminal end presents a certain rigidity and explores alfa helix conformations. This prefolded structure could be essential for binding to the substrate and folding. On the binding and folding mechanism we could obtain evidence that suggests the existence of an intermediate complex between the free unfolded form and the RNA bound folded form. In this intermediate complex the protein is also mainly unfolded but bound to the substrate. Finally, on a third stage of this work we performed a structural study of full-length micro-RNA precursors. We have focused on validating the determinants that allow the recognition of the micro-RNA position in the precursor by the processing complex involving DCL1 and the accessory proteins HYL1 and SERRATE. Employing biochemical techniques for probing secondary structure and strand flexibility we have studied four precursors from different families. We were able to experimentally validate structural determinants proposed on the basis of in silico calculated structures. We found evidence that suggests the position of the first cleavage site, which is essential for defining the register for the successive DCL1 cuts, could be related with an abrupt change from a flexible region of the strand to a much lesser flexible one. This peculiarity could be allowing for a specific interaction with the proteins involved in the processing complex.
Descripción
Palabras clave
RMN, MiARN, IDP, Resonancia magnética nuclear, Proteína intrínsecamente desordenada