TY - JOUR
T1 - Requirements for gene silencing mediated by U1 snRNA binding to a target sequence
AU - Abad, Xabi
AU - Vera, Maria
AU - Jung, Stephen P.
AU - Oswald, Evelyn
AU - Romero, Inés
AU - Amin, Vaibhav
AU - Fortes, Puri
AU - Gunderson, Samuel J.
N1 - Funding Information:
We thank Dr Peter G. Stockley and Gabriella Basnak for anti-MS2 antibody. Technical assistance by N. Razquin is thankfully acknowledged. This work was supported by CICYT (SAF2003-01804), CICYT (BIO2006-13225) and through the ‘UTE project CIMA’ to P.F. as well as by NIH grant GM057286 to S.I.G. Funding to pay the Open Access publication charges for this article were provided by the grants listed above.
PY - 2008/4
Y1 - 2008/4
N2 - 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.
AB - 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.
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U2 - 10.1093/nar/gkn068
DO - 10.1093/nar/gkn068
M3 - Article
C2 - 18299285
AN - SCOPUS:42449160202
SN - 0305-1048
VL - 36
SP - 2338
EP - 2352
JO - Nucleic acids research
JF - Nucleic acids research
IS - 7
ER -