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

Xabi Abad, Maria Vera Ugalde, Stephen P. Jung, Evelyn Oswald, Inés Romero, Vaibhav Amin, Puri Fortes, Samuel J. Gunderson

Research output: Contribution to journalArticle

35 Citations (Scopus)

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 (≤ 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.

Original languageEnglish (US)
Pages (from-to)2338-2352
Number of pages15
JournalNucleic Acids Research
Volume36
Issue number7
DOIs
StatePublished - Apr 2008
Externally publishedYes

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Gene Silencing
U1 Small Nuclear Ribonucleoproteins
RNA Splice Sites
Polyadenylation
RNA Precursors
Hydrogen Bonding
Mutagenesis
Base Pairing
Serine
Arginine
Exons
RNA
Gene Expression
Genes
U1 small nuclear RNA
RNA Splicing Factors

ASJC Scopus subject areas

  • Genetics

Cite this

Abad, X., Vera Ugalde, M., Jung, S. P., Oswald, E., Romero, I., Amin, V., ... Gunderson, S. J. (2008). Requirements for gene silencing mediated by U1 snRNA binding to a target sequence. Nucleic Acids Research, 36(7), 2338-2352. https://doi.org/10.1093/nar/gkn068

Requirements for gene silencing mediated by U1 snRNA binding to a target sequence. / Abad, Xabi; Vera Ugalde, Maria; Jung, Stephen P.; Oswald, Evelyn; Romero, Inés; Amin, Vaibhav; Fortes, Puri; Gunderson, Samuel J.

In: Nucleic Acids Research, Vol. 36, No. 7, 04.2008, p. 2338-2352.

Research output: Contribution to journalArticle

Abad, X, Vera Ugalde, M, Jung, SP, Oswald, E, Romero, I, Amin, V, Fortes, P & Gunderson, SJ 2008, 'Requirements for gene silencing mediated by U1 snRNA binding to a target sequence', Nucleic Acids Research, vol. 36, no. 7, pp. 2338-2352. https://doi.org/10.1093/nar/gkn068
Abad X, Vera Ugalde 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 Research. 2008 Apr;36(7):2338-2352. https://doi.org/10.1093/nar/gkn068
Abad, Xabi ; Vera Ugalde, Maria ; Jung, Stephen P. ; Oswald, Evelyn ; Romero, Inés ; Amin, Vaibhav ; Fortes, Puri ; Gunderson, Samuel J. / Requirements for gene silencing mediated by U1 snRNA binding to a target sequence. In: Nucleic Acids Research. 2008 ; Vol. 36, No. 7. pp. 2338-2352.
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