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Nucleic Acids Research Advance Access published online on February 24, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn068
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

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

Xabi Abad1, Maria Vera1, Stephen P. Jung2, Evelyn Oswald2, Inés Romero1, Vaibhav Amin2, Puri Fortes1,* and Samuel I. Gunderson2,*

1Division of Hepatology and Gene Therapy, CIMA/UNAV. Pio XII, 55, 31008 Pamplona, Spain and 2Rutgers University, 604 Allison Road, Piscataway, NJ 08854, USA

*To whom correspondence should be addressed. Tel: +34 948 194700, ext. 4025; Fax: 34 948 194717; Email: pfortes{at}unav.es Correspondence may also be addressed to Samuel I. Gunderson. Tel: +732 445 1016; Fax: +732 445 4213; Email: gunderson{at}biology.rutgers.edu

Received December 19, 2007. Revised February 4, 2008. Accepted February 4, 2008.

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.

Present address: Maria Vera, Department of Biochemistry, New York University School of Medicine, NY 10016, USA.

The authors wish it to be known that the seventh and the eighth authors should be regarded as joint Last Authors.


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