Inhibiting expression of specific genes in mammalian cells with 5′ end-mutated U1 small nuclear RNAs targeted to terminal exons of pre-mRNA
Inhibiting expression of specific genes in mammalian cells with 5′ end-mutated U1 small nuclear RNAs targeted to terminal exons of pre-mRNA
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DOI：
10.1073/pnas.1332669100
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发表时间：
2003-07-08
影响因子：
11.1
通讯作者：
Gunderson, SI
中科院分区：
文献类型：
--
作者：
Fortes, P;Cuevas, Y;Gunderson, SI
减少或消除给定基因的表达可能需要多种方法来确保覆盖给定哺乳动物细胞中的所有基因。我们和其他人[Furth，P.A.，Choe，W. T.，雷克斯，J.H.，Byrne，J. C.，和Baker，C. C.（1994）Mol. Cell. Biol.14，5278-5289]先前已经表明，天然或具有5'端突变的U1小核（sn）RNA可以特异性抑制哺乳动物细胞中的报道基因表达。当U1 snRNA 5'端碱基对靠近报告基因前mRNA的多聚腺苷酸化信号时，会发生这种抑制。这种碱基配对抑制了poly（A）尾的添加，这是mRNA生物合成中的一个关键的、几乎普遍的步骤，导致mRNA的降解。在这里，我们证明，内源性哺乳动物基因的表达可以有效地抑制瞬时或稳定表达的5'端突变的U1 snRNA。此外，我们确定了抑制机制，并建立了一套规则，使用这种技术，以提高抑制的效率。两个U1 snRNA碱基配对到一个单一的前mRNA协同作用，导致高达700倍的抑制特异性报告基因的表达和25倍的抑制内源性基因。令人惊讶的是，从U1 snRNA结合位点到poly（A）信号的距离对于抑制并不关键，相反，U1 snRNA必须靶向前体mRNA的末端外显子。这可能反映了5'端突变的U1 snRNA对外显子定义模型所描述的末端外显子定义的破坏。
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.