Determinación de la interacción entre los RNAs mitocondriales no codificantes y proteínas con dominio de unión a RNA de doble hebra
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Fecha
2014
Profesor/a Guía
Idioma
es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
Nuestro laboratorio identificó una familia de RNAs no codificantes mitocondriales (ncmtRNAs), conformada por transcritos sentido (SncmtRNA) y antisentido (ASncmtRNA), los que están formados por el mtrRNA 16s sentido y antisentido,
respectivamente, con un repetido invertido unido covalentemente al extremo 5'. En consecuencia, estos RNAs forman estructuras tallo-asa. Por hibridación in situ, la detección de los ncmtRNAs se relaciona con el estado proliferativo de las células,encontrándose tres fenotipos: 1) en células normales proliferativas se detectan ambos transcritos, 2) en células tumorales solo se detecta el transcrito sentido con una baja detección de los ASncmtRNAs y 3) en células normales no proliferativas no se detecta ninguno de ellos. La interferencia de los ASncmtRNAs induce la muerte selectiva de células tumorales, no así de células normales. Para indagar sobre las funciones de éstos transcritos para así comprender los mecanismos involucrados en los efectos de la interferencia de los ASncmtRNAs, exploramos
posibles proteínas que interactúan con ellos en la célula. El análisis de la región de RNA de doble hebra (dsRNA) nos permite considerar una interacción con proteínas que poseen dominios de unión a dsRNA (DRBP). Para este trabajo
elegimos como candidatas proteínas involucradas en la vía de microRNA (miRNA) y, adicionalmente, PKR, involucrada en respuesta antiviral frente a dsRNA. Nuestros resultados demuestran la formación de complejos entre el SncmtRNA
sintetizado in vitro y proteínas(s), mediante ensayo de cambio en la movilidad electroforética (EMSA). Además, ensayos de inmunoprecipitación a partir de lisados de células SK-MEL-2 (melanoma) y melanocitos humanos normales,
muestran una asociación de los ncmtRNAs con DROSHA, DICER, AGO-2 y ADAR-1, sugiriendo un rol de estos transcritos en la vía de RNAi. Por otro lado, la interacción con PKR sugiere la participación de éstos RNAs en inmunidad innata celular. Los resultados obtenidos en esta tesis contribuyen al entendimiento de las funciones de los ncmtRNAs y ayudarán a la aplicación de la interferencia de los ASncmtRNAs para el desarrollo de una terapia antitumoral selectiva y eficaz.
Our group described a family of human non-coding mitochondrial RNAs (ncmtRNAs) consisting of Sense (SncmtRNA) and Antisense (ASncmtRNA) transcripts, which contain inverted repeats joined to the 5 · end of the sen se or antisense 16S mitochondrial rRNA, respectively. Consequently, these transcripts form stem-loop structures. Experimental evidence shows that the expression of these transcripts is related to the proliferative status of cells, distinguishing three distinct phenotypes by in situ hybridization. 1) Normal proliferating cells express both , 2) tumor cells express the SncmtRNA but down-regulate the ASncmtRNA and 3) Normal resting cells express neither of these transcripts. The interference of ASncmtRNAs with the complementary oligonucleotides induces selective death of tumor cells without affecting normal cells. To investigate the functions of these transcripts and understand the mechanism involved in the effects caused by the interference of ASncmtRNAs, we explored possible proteins that interact with these transcripts in the cel l. Analysis of the double-stranded RNA region of these transcripts led us to consider an interaction with proteins that possess dsRNA binding domains (DRBPs). DRBPs have specificity for stem-loop structures of many RNAs, and comprise proteins involved in diverse cellular mechanisms. The candidates of DRBPs that could interact with the ncmtRNAs are proteins involved in the microRNA (miRNA) pathway such as Dicer, Drosha, Argonaute-2 and ADAR1 , and the PKR protein, involved in antiviral response . Our results indicate that there are RNA-protein complexes by electrophoretic mobility shift assay (EMSA), using in vitro transcribed SncmtRNA. Furthermore, immunoprecipitations assays using SK-MEL-2 and HEMn cell lysates show an association of ncmtRNAs with DICER, DROSHA, AG0-2 and ADAR-1 , suggesting a role for these transcripts in the RNAi pathway. Furthermore, interaction with PKR suggests an involvement of these RNAs in innate immunity. The results obtained here will help to understand the functions of ncmtRNAs and will assist the implementation of the interference ASncmtRNAs for the development an effective antitumor therapy.
Our group described a family of human non-coding mitochondrial RNAs (ncmtRNAs) consisting of Sense (SncmtRNA) and Antisense (ASncmtRNA) transcripts, which contain inverted repeats joined to the 5 · end of the sen se or antisense 16S mitochondrial rRNA, respectively. Consequently, these transcripts form stem-loop structures. Experimental evidence shows that the expression of these transcripts is related to the proliferative status of cells, distinguishing three distinct phenotypes by in situ hybridization. 1) Normal proliferating cells express both , 2) tumor cells express the SncmtRNA but down-regulate the ASncmtRNA and 3) Normal resting cells express neither of these transcripts. The interference of ASncmtRNAs with the complementary oligonucleotides induces selective death of tumor cells without affecting normal cells. To investigate the functions of these transcripts and understand the mechanism involved in the effects caused by the interference of ASncmtRNAs, we explored possible proteins that interact with these transcripts in the cel l. Analysis of the double-stranded RNA region of these transcripts led us to consider an interaction with proteins that possess dsRNA binding domains (DRBPs). DRBPs have specificity for stem-loop structures of many RNAs, and comprise proteins involved in diverse cellular mechanisms. The candidates of DRBPs that could interact with the ncmtRNAs are proteins involved in the microRNA (miRNA) pathway such as Dicer, Drosha, Argonaute-2 and ADAR1 , and the PKR protein, involved in antiviral response . Our results indicate that there are RNA-protein complexes by electrophoretic mobility shift assay (EMSA), using in vitro transcribed SncmtRNA. Furthermore, immunoprecipitations assays using SK-MEL-2 and HEMn cell lysates show an association of ncmtRNAs with DICER, DROSHA, AG0-2 and ADAR-1 , suggesting a role for these transcripts in the RNAi pathway. Furthermore, interaction with PKR suggests an involvement of these RNAs in innate immunity. The results obtained here will help to understand the functions of ncmtRNAs and will assist the implementation of the interference ASncmtRNAs for the development an effective antitumor therapy.
Notas
Tesis (Magíster en Biotecnología)
Palabras clave
Proteínas Mitocondriales, Transcripción Genética.