?Estudio de la funcionalidad de la prote?na del s?ndrome de Werner (WRN) en la maquinaria traduccional y alteraciones moleculares derivadas de su silenciamiento en c?lulas HeLa?

Abstract

El presente trabajo tuvo como finalidad evaluar la relaci?n funcional de la prote?na del S?ndrome de Werner (WRN) con la maquinaria traduccional en c?lulas HeLa depletadas para WRN. Se utiliz? el sistema lentiviral inducible en c?lulas HeLa para desregular la expresi?n de WRN y realizar los posteriores an?lisis de qPCR y Western Blot. As? mismo, se utiliz? el sistema de expresi?n de baculovirus y el sistema de transcripci?n/traducci?n para expresar la prote?na WRN. Finalmente, se realizaron los ensayos de traducci?n in vitro utilizando vectores reporteros. Se localiz? la presencia de la prote?na WRN en la fracci?n citoplasm?tica y polisomal. Adem?s, no se encontraron diferencias entre la expresi?n de los ARNm de las prote?nas ribosomales RPS6, RPL7a entre las c?lulas HeLa carentes de WRN y c?lulas HeLa control. Sin embargo, se encontr? un aumento de ~3 veces (326%) m?s de la expresi?n de los ARNm de IDH1 en c?lulas HeLa debido quiz?s al estr?s celular. Adicionalmente, los ensayos de traducci?n in vitro entre c?lulas HeLa depletadas para WRN y c?lulas control, lograron determinar que la ausencia de WRN da como resultado una disminuci?n en la traducci?n de prote?nas (65% menos con respecto al control) y con el uso del inhibidor de la actividad helicasa de WRN NSC-19630 (67.5% menos con respecto al control). La depleci?n de WRN no altera la expresi?n de los ARNm y las prote?nas ribosomales RPS3, RPS6 y RPL7A. As? mismo, se demostr? que la prote?na de WRN presenta una funci?n en la traducci?n de prote?nas al comparar los niveles de traducci?n de prote?nas reporteras en c?lulas HeLa shWRN y shCTR, y mediante la inhibici?n de la actividad helicasa de WRN. The aim of this work was to evaluate the functional relationship of the Werner Syndrome protein (WRN) in the translational machinery in HeLa cells depleted for WRN. The inducible lentiviral system was used in HeLa cells to downregulate the expression of WRN and perform the subsequent analyzes of qPCR and Western Blot. Likewise, the baculovirus expression system and the transcription/translation system were used to express the WRN protein. Finally, the in vitro translation assays were carried out using reporter vectors. The WRN protein was located in the cytoplasmic and polysomal fraction. No differences were found between the expression of mRNA levels of RPS6, RPL7a between HeLa shWRN and HeLa control cells. However, a ~ 3 fold (326%) increase in the metabolic gene IDH1 in HeLa cells was found due to cell stress. Moreover, the in vitro translation assays between shWRN HeLa and control were able to determine that the absence of WRN results in a decrease in the translation of proteins (65% less compared to the control) and with the use of the helicase activity inhibitor of WRN (NSC-19630) (67.5% less compared to the control). WRN depletion does not alter the expression of mRNAs and ribosomal proteins RPS3, RPS6 and RPL7A. Furthermore, it was demonstrated that the WRN protein has a function in the translation of proteins when comparing the levels of translation of reporter proteins in HeLa cells shWRN and shCTR, and by inhibiting the helicase activity of WRN.