Requirements for gene silencing mediated by U1 snRNA binding to a target sequence

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%) an...

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Detalles Bibliográficos
Autores: Abad, X. (Xabier)|||/items/050ca3bc-2ff5-40d8-a24c-72cb58605954, Vera, M. (María)|||/items/8f8ce09a-134e-4bcb-9c35-f04d2477d385, Jung, S.P. (Stephen P.)|||/items/ecafeb73-8006-4fac-96be-63d52a57f045, Oswald, E. (Evelyn)|||/items/ee92baad-dfdd-4873-9ee4-c02a754652c1, Romero, I. (Inés)|||/items/151064ed-30d8-4245-a50b-2d325e080932, Amin, V. (Vaibhav)|||/items/36146211-e32a-4855-be4c-e80703b6358c, Fortes, P. (Puri)|||/items/4b719a39-983d-402a-bb2a-2bdd276be18e, Gunderson, S.I. (Samuel I.)|||/items/a4e15896-51e7-4d3e-a116-15d28e23501f
Tipo de recurso: artículo
Fecha de publicación:2008
País:España
Institución:Universidad de Navarra
Repositorio:Dadun. Depósito Académico Digital de la Universidad de Navarra
Idioma:inglés
OAI Identifier:oai:dadun.unav.edu:10171/23171
Acceso en línea:https://hdl.handle.net/10171/23171
Access Level:acceso abierto
Palabra clave:RNA Interference
RNA Precursors/chemistry
RNA, Messenger/chemistry
RNA, Small Nuclear/chemistry
Descripción
Sumario: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.

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