Tesis doctoral de Oscar Aparicio Ruiz
Little is known about shrna in vivo processing, accumulation, functional kinetics, and side effects related to shrna saturation of the cellular gene silencing machinery. Therefore, we constructed first-generation recombinant adenoviruses encoding different shrnas against murine atp-binding cassette multidrug resistance protein 2 (abcc2), which is involved in liver transport of bilirubin to bile, and analyzed abcc2 silencing kinetics. C57/bl6 mice injected with these viruses showed significant impairment of abcc2 function for up to 3 weeks, as reflected by increased serum bilirubin levels. The lack of abcc2 function correlated with a specific reduction of abcc2 mrna and with high levels of processed shrnas targeting abcc2. Inhibition was lost at longer times postinfection, correlating with a decrease in the accumulation of processed shrnas. This finding suggests that a minimal amount of processed shrnas is required for efficient silencing in vivo. This system was also used to evaluate the effect of shrna expression on the saturation of silencing factors. Saturation of the cellular silencing processing machinery alters the accumulation and functionality of endogenous micrornas (mirnas) and pre-mirnas. However, expression of functional exogenous shrnas did not change the levels of endogenous mirnas or their precursors. In summary, that adenoviral vectors can deliver sufficient shrnas to mediate inhibition of gene expression without saturating the silencing machinery. Posttranscriptional gene silencing allows sequence-specific control of gene expression. Specificity is guaranteed by small antisense rnas such as micrornas (mirnas) or small interfering rnas (sirnas). Functional mirnas derive from longer double-stranded rna (dsrna) molecules that are cleaved to pre-mirnas in the nucleus and are transported by exportin 5 (exp 5) to the cytoplasm. Adenovirus-infected cells express virus-associated (va) rnas, which are dsrna molecules similar in structure to pre-mirnas. Va rnas are also transported by exp 5 to the cytoplasm, where they accumulate. Here we show that small rnas derived from va rnas (svarnas), similar to mirnas, can be found in adenovirus-infected cells. Va rna processing to mivarnas requires neither viral replication nor viral protein expression, as evidenced by the fact that mivarna accumulation can be detected in cells transfected with va sequences. Mivarnas are efficiently bound by argonaute 2, the endonuclease of the rna-induced silencing complex, and behave as functional sirnas, in that they inhibit the expression of reporter genes with complementary sequences. Blocking mivarna-mediated inhibition affects efficient adenovirus production, indicating that mivarnas are required for virus viability. Mivarnas are important for virus production, suggesting that they may target cellular or viral genes that affect virus viability. We first identified genes downregulated in the presence of va rnas by microarray analysis. These genes were then screened for mivarna target sites using several bioinformatic tools. Thus, combination of microarray analysis and bioinformatics allowed us to identify the splicing and translation regulator tia-1 as a putative mivarna target, we show that tia-1 is downregulated at mrna and protein levels in infected cells expressing functional mivarnas and in transfected cells that express mivarnai-138, one of the more abundant viral mirnas. Also, reporter assays show that tia-1 is downregulated directly by mivarnai-138. Finally, we show that tia-1 expression increases accumulation of e1a, a viral transcription factor whose levels are decreased at late times post-infection. These results suggest that tia-1 expression decreased by mivarnai-138 could reduce e1a levels late in infection and therefore, that mivarnai-138-mediated downregulation of tia-1 could contribute to the early to late shift of adenovirus infection.
Datos académicos de la tesis doctoral «Identificación y análisis del efecto celular de rnas pequeños expresados a partir de adenovirus«
- Título de la tesis: Identificación y análisis del efecto celular de rnas pequeños expresados a partir de adenovirus
- Autor: Oscar Aparicio Ruiz
- Universidad: Navarra
- Fecha de lectura de la tesis: 24/06/2008
Dirección y tribunal
- Director de la tesis
- María Purificacion Fortes Alonso
- Tribunal
- Presidente del tribunal: Juan Ortin monton
- Miguel angel Barajas velez (vocal)
- james Sutherland (vocal)
- ramon Alemany bonastre (vocal)