Análisis del procesamiento del arn mensajero de AtbZIP60 bajo distintos tratamientos que activan la respuesta a la acumulación de proteínas mal plegadas en Arabidopsis thaliana usando electroforesis capilar
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2015
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es
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Universidad Andrés Bello
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Licencia CC
Resumen
La respuesta a proteínas mal plegada (UPR) es una vía de señalización asociada al retículo endoplásmico (ER). Normalmente, las proteínas sintetizadas en el ER que no logran adquirir su conformación nativa se degradan por un mecanismo denominado degradación asociada a retículo (ERAD). Sin embargo, si este mecanismo falla en degradar proteínas no plegadas / mal plegadas, éstas son retenidas en el ER activando la UPR. Durante este proceso un conjunto de genes que pueden ayudar al plegamiento correcto de las proteínas son regulados positivamente a nivel transcripcional y traduccional. Estudios recientes sobre Arabidopsis thaliana y arroz identificaron una vía de señalización en las cual una proteína transmembrana de ER llamada IRE1 cataliza el empalme citoplasmático y no convencional de un ARN mensajero conduciendo a la síntesis de un factor de transcripción activo capaz de activar los genes de respuesta a UPR. Este factor de transcripción se conoce como bZIP60 en Arabidopsis y bZIP50 en arroz. Informes recientes han mostrado que dos agentes químicos clásicos utilizados para inducir la UPR (tunicamicina y DTT) activan el procesamiento de bZIP60. Sin embargo, la dinámica y la temporalidad del empalme no convencional son desconocidas. Adicionalmente, condiciones fisiológicas en la planta como la acumulación de la fitohormona, ácido salicílico (SA) y estrés por calor; también pueden inducir el procesamiento de bZIP60. En dichas condiciones, bZIP60 procesado, es apenas detectable al utilizar electroforesis en geles de agarosa como instrumento analítico. En esta tesis se propone que el empalme no convencional de bZIP60 podría ser analizado mediante electroforesis capilar, debido a su capacidad de alta resolución y posible cuantificación. Como una prueba de concepto, se analizó la dinámica y temporalidad del procesamiento del gen bZIP60 en las plantas tratadas con tunicamicina, DTT, SA y calor. Los resultados indican que el procesamiento de AtbZIP60 es un proceso dinámico y su ocurrencia y la magnitud es estímulo-dependiente. Además, el procesamiento AtbZIP60 en diferentes tejidos de plantas con o sin estrés, sugiere que la activación del UPR es un proceso tejido específico. Por otra parte, el análisis en plantas mutantes que carecen de otros componentes de la señalización de la UPR, indican que la rama IRE1 / AtbZIP60 no compensa la ausencia de otros componentes tales como bZIP28 bajo condiciones de estrés de ER. En su conjunto los resultados sugieren que el procesamiento de AtbZIP60 es un proceso altamente regulado y que eso no depende únicamente de los estímulos, sino más bien de cómo cada tejido percibe dichos estímulos.
The unfolded protein response (UPR) is a signaling pathway associated with the endoplasmic reticulum (ER). Normally, proteins synthetized on the ER that fail to acquire their native conformation are degraded by a mechanism termed ER-associated degradation (ERAD). Nevertheless, if this mechanism fails to degrade unfolded/misfolded proteins, these are retained in the ER triggering the UPR. During this process a set of genes that can assist the correct folding of proteins are up regulated at transcriptional and translational level. Recent studies on Arabidopsis thaliana and rice identified a signaling pathway in which an ER transmembrane protein called IRE1 catalyzed the unconventional cytoplasmic splicing of a transcription factor mRNA leading to the synthesis of an active transcription factor capable of activating the UPR-responding genes. This transcription factor is known as bZIP60 in Arabidopsis and bZIP50 in rice. Recent reports have shown that two classical chemicals used to induce the UPR (tunicamycin and DTT) activate the processing of bZIP60. Nevertheless the dynamics and temporality of the unconventional splicing is unknown. Additionally, physiological conditions on the plant such as the accumulation of the plant hormone salicylic acid (SA) and heat stress can also induce the processing of bZIP60. In these conditions, bZIP60 processed is barely detectable using agarose gel electrophoresis as an analytical tool. In this thesis we propose that the non-conventional splicing of bZIP60 could be analyzed using capillary electrophoresis, due to its high resolution capability and possible quantification. As a proof of concept the dynamic and temporal processing of bZIP60 gene was analyzed in plants treated with tunicamycin, DTT, SA and heat. The results indicate that the processing of AtbZIP60 is a dynamic process and its ocurrence and magnitude is stimulus-dependent. In addition, AtbZIP60 processing in different plant tissues with or without stress suggests that UPR activation is a tissue specific process. On the other hand, analyses on mutants that lack other components of the UPR pathway indicate that the IRE1/AtbZIP60 branch of the UPR does not compensate the absence of other components such as bZIP28 under ER stress conditions. Taken together, the results suggest that the processing of AtbZIP60 is a highly regulated process that that does not depend solely on stimuli but rather how each tissue perceive these stimuli.
The unfolded protein response (UPR) is a signaling pathway associated with the endoplasmic reticulum (ER). Normally, proteins synthetized on the ER that fail to acquire their native conformation are degraded by a mechanism termed ER-associated degradation (ERAD). Nevertheless, if this mechanism fails to degrade unfolded/misfolded proteins, these are retained in the ER triggering the UPR. During this process a set of genes that can assist the correct folding of proteins are up regulated at transcriptional and translational level. Recent studies on Arabidopsis thaliana and rice identified a signaling pathway in which an ER transmembrane protein called IRE1 catalyzed the unconventional cytoplasmic splicing of a transcription factor mRNA leading to the synthesis of an active transcription factor capable of activating the UPR-responding genes. This transcription factor is known as bZIP60 in Arabidopsis and bZIP50 in rice. Recent reports have shown that two classical chemicals used to induce the UPR (tunicamycin and DTT) activate the processing of bZIP60. Nevertheless the dynamics and temporality of the unconventional splicing is unknown. Additionally, physiological conditions on the plant such as the accumulation of the plant hormone salicylic acid (SA) and heat stress can also induce the processing of bZIP60. In these conditions, bZIP60 processed is barely detectable using agarose gel electrophoresis as an analytical tool. In this thesis we propose that the non-conventional splicing of bZIP60 could be analyzed using capillary electrophoresis, due to its high resolution capability and possible quantification. As a proof of concept the dynamic and temporal processing of bZIP60 gene was analyzed in plants treated with tunicamycin, DTT, SA and heat. The results indicate that the processing of AtbZIP60 is a dynamic process and its ocurrence and magnitude is stimulus-dependent. In addition, AtbZIP60 processing in different plant tissues with or without stress suggests that UPR activation is a tissue specific process. On the other hand, analyses on mutants that lack other components of the UPR pathway indicate that the IRE1/AtbZIP60 branch of the UPR does not compensate the absence of other components such as bZIP28 under ER stress conditions. Taken together, the results suggest that the processing of AtbZIP60 is a highly regulated process that that does not depend solely on stimuli but rather how each tissue perceive these stimuli.
Notas
Tesis (Magíster en Biotecnología)
Proyecto FONDECYT No1110954.
Proyecto FONDECYT No1110954.
Palabras clave
Genes, Arabidopsis Thaliana