In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 100, No. 14 ( 2003-07-08), p. 8264-8269
Abstract:
Reducing or eliminating expression of a given gene is likely to require
multiple methods to ensure coverage of all of the genes in a given mammalian cell. We and others [Furth, P. A., Choe, W. T., Rex, J. H., Byrne, J. C., and
Baker, C. C. (1994) Mol. Cell. Biol. 14, 5278–5289] have previously shown that U1 small nuclear (sn) RNA, both natural or with 5′
end mutations, can specifically inhibit reporter gene expression in mammalian cells. This inhibition occurs when the U1 snRNA 5′ end base pairs near
the polyadenylation signal of the reporter gene's pre-mRNA. This base pairing inhibits poly(A) tail addition, a key, nearly universal step in mRNA
biosynthesis, resulting in degradation of the mRNA. Here we demonstrate that expression of endogenous mammalian genes can be efficiently inhibited by
transiently or stably expressed 5′ end-mutated U1 snRNA. Also, we determine the inhibitory mechanism and establish a set of rules to use this
technique and to improve the efficiency of inhibition. Two U1 snRNAs base paired to a single pre-mRNA act synergistically, resulting in up to 700-fold
inhibition of the expression of specific reporter genes and 25-fold inhibition of endogenous genes. Surprisingly, distance from the U1 snRNA binding site to
the poly(A) signal is not critical for inhibition, instead the U1 snRNA must be targeted to the terminal exon of the pre-mRNA. This could reflect a
disruption by the 5′ end-mutated U1 snRNA of the definition of the terminal exon as described by the exon definition model.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1332669100
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2003
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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