Abad, X. (Xabier)
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- Requirements for gene silencing mediated by U1 snRNA binding to a target sequence(Oxford University Press, 2008) Oswald, E. (Evelyn); Amin, V. (Vaibhav); Vera, M. (María); Gunderson, S.I. (Samuel I.); Abad, X. (Xabier); Fortes, P. (Puri); Jung, S.P. (Stephen P.); Romero, I. (Inés)U1 interference (U1i) is a novel method to block gene expression. U1i requires expression of a 5'-end-mutated U1 snRNA designed to base pair to the 3'-terminal exon of the target gene's pre-mRNA that leads to inhibition of polyadenylation. Here, we show U1i is robust (> or =95%) and a 10-nt target length is sufficient for good silencing. Surprisingly, longer U1 snRNAs, which could increase annealing to the target, fail to improve silencing. Extensive mutagenesis of the 10-bp U1 snRNA:target duplex shows that any single mismatch different from GU at positions 3-8, destroys silencing. However, mismatches within the other positions give partial silencing, suggesting that off-target inhibition could occur. The specificity of U1i may be enhanced, however, by the fact that silencing is impaired by RNA secondary structure or by splicing factors binding nearby, the latter mediated by Arginine-Serine (RS) domains. U1i inhibition can be reconstituted in vivo by tethering of RS domains of U1-70K and U2AF65. These results help to: (i) define good target sites for U1i; (ii) identify and understand natural cellular examples of U1i; (iii) clarify the contribution of hydrogen bonding to U1i and to U1 snRNP binding to 5' splice sites and (iv) understand the mechanism of U1i.
- Adenovirus VA RNA-derived miRNAs target cellular genes involved in cell growth, gene expression and DNA repair(Oxford University Press, 2010) Aparicio, O. (Óscar); Guruceaga, E. (Elizabeth); Razquin, N. (Nerea); Abad, X. (Xabier); Fortes, P. (Puri); Carnero, E. (Elena); Segura, V. (Víctor)Adenovirus virus-associated (VA) RNAs are processed to functional viral miRNAs or mivaRNAs. mivaRNAs are important for virus production, suggesting that they may target cellular or viral genes that affect the virus cell cycle. To look for cellular targets of mivaRNAs, 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. The combination of microarray analysis and bioinformatics allowed us to select 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 most abundant adenoviral miRNAs. Also, reporter assays show that TIA-1 is downregulated directly by mivaRNAI-138. To determine whether mivaRNAs could target other cellular genes we analyzed 50 additional putative targets. Thirty of them were downregulated in infected or transfected cells expressing mivaRNAs. Some of these genes are important for cell growth, transcription, RNA metabolism and DNA repair. We believe that a mivaRNA-mediated fine tune of the expression of some of these genes could be important in adenovirus cell cycle.
- Increased in vivo inhibition of gene expression by combining RNA interference and U1 inhibition(Oxford University Press, 2012) Gonzalez-Rojas, S.J. (Sandra Jovanna); Blazquez, L. (Lorea); Abad, A. (Amaya); Razquin, N. (Nerea); Abad, X. (Xabier); Fortes, P. (Puri)Inhibition of gene expression can be achieved with RNA interference (RNAi) or U1 small nuclear RNA-snRNA-interference (U1i). U1i is based on U1 inhibitors (U1in), U1 snRNA molecules modified to inhibit polyadenylation of a target pre-mRNA. In culture, we have shown that the combination of RNAi and U1i results in stronger inhibition of reporter or endogenous genes than that obtained using either of the techniques alone. We have now used these techniques to inhibit gene expression in mice. We show that U1ins can induce strong inhibition of the expression of target genes in vivo. Furthermore, combining U1i and RNAi results in synergistic inhibitions also in mice. This is shown for the inhibition of hepatitis B virus (HBV) sequences or endogenous Notch1. Surprisingly, inhibition obtained by combining a U1in and a RNAi mediator is higher than that obtained by combining two U1ins or two RNAi mediators. Our results suggest that RNAi and U1i cooperate by unknown mechanisms to result in synergistic inhibitions. Analysis of toxicity and specificity indicates that expression of U1i inhibitors is safe. Therefore, we believe that the combination of RNAi and U1i will be a good option to block damaging endogenous genes, HBV and other infectious agents in vivo.
- Combination of RNA interference and U1 inhibition leads to increased inhibition of gene expression(Oxford University Press, 2010) Abad, A. (Amaya); Razquin, N. (Nerea); Abad, X. (Xabier); Fortes, P. (Puri)RNA interference (RNAi) has been revolutionary for the specific inhibition of gene expression. However, the application of RNAi has been hampered by the fact that many siRNAs induce dose-dependent unwanted secondary effects. Therefore, new methods to increase inhibition of gene expression with low doses of inhibitors are required. We have tested the combination of RNAi and U1i (U1 small nuclear RNA--snRNA--interference). U1i is based on U1 inhibitors (U1in), U1 snRNA molecules modified to target a pre-mRNA and inhibit its gene expression by blocking nuclear polyadenylation. The combination of RNAi and U1i resulted in stronger inhibition of reporter or endogenous genes than that obtained using either of the techniques alone. The increased inhibition observed is stable over time and allows higher inhibition than the best obtained with either of the inhibitors alone even with decreased doses of the inhibitors. We believe that the combination of RNAi and U1i will be of interest when higher inhibition is required or when potent inhibitors are not available. Also, the combination of these techniques would allow functional inhibition with a decreased dose of inhibitors, avoiding toxicity due to dose-dependent unwanted effects.