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Dadun > Depósito Académico > CIMA (Centro de Investigación Médica Aplicada) > Área de Terapia génica y Hepatología > Vectores > DA - CIMA - Terapia génica y Hepatología - Vectores - Artículos de revista >

Requirements for gene silencing mediated by U1 snRNA binding to a target sequence
Authors: Abad, X. (Xabier)
Vera, M. (María)
Jung, S.P. (Stephen P.)
Oswald, E. (Evelyn)
Romero, I. (Inés)
Amin, V. (Vaibhav)
Fortes, P. (Puri)
Gunderson, S.I. (Samuel I.)
Keywords: RNA Interference
RNA Precursors/chemistry
RNA, Messenger/chemistry
RNA, Small Nuclear/chemistry
Issue Date: 2008
Publisher: Oxford University Press
Publisher version: http://nar.oxfordjournals.org/content/36/7/2338
ISSN: 1362-4962
Citation: Abad X, Vera M, Jung SP, Oswald E, Romero I, Amin V, et al. Requirements for gene silencing mediated by U1 snRNA binding to a target sequence. Nucleic Acids Res 2008 Apr;36(7):2338-2352.
Abstract
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.
Permanent link: http://hdl.handle.net/10171/23171
Appears in Collections:DA - CIMA - Terapia génica y Hepatología - Vectores - Artículos de revista

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