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首页> 外文期刊>Molecular and Cellular Biology >U1 small nuclear RNAs with altered specificity can be stably expressed in mammalian cells and promote permanent changes in pre-mRNA splicing.
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U1 small nuclear RNAs with altered specificity can be stably expressed in mammalian cells and promote permanent changes in pre-mRNA splicing.

机译：具有改变的特异性的U1小核RNA可以在哺乳动物细胞中稳定表达，并促进前mRNA剪接中的永久性变化。

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摘要

Pre-mRNA 5' splice site activity depends, at least in part, on base complementarity to U1 small nuclear RNA. In transient coexpression assays, defective 5' splice sites can regain activity in the presence of U1 carrying compensatory changes, but it is unclear whether such mutant U1 RNAs can be permanently expressed in mammalian cells. We have explored this issue to determine whether U1 small nuclear RNAs with altered specificity may be of value to rescue targeted mutant genes or alter pre-mRNA processing profiles. This effort was initiated following our observation that U1 with specificity for a splice site associated with an alternative H-ras exon substantially reduced the synthesis of the potentially oncogenic p21ras protein in transient assays. We describe the development of a mammalian complementation system that selects for removal of a splicing-defective intron placed within a drug resistance gene. Complementation was observed in proportion to the degree of complementarity between transfected mutant U1 genes and different defective splice sites, and all cells selected in this manner were found to express mutant U1 RNA. In addition, these cells showed specific activation of defective splice sites presented by an unlinked reporter gene. We discuss the prospects of this approach to permanently alter the expression of targeted genes in mammalian cells.
机译：mRNA前5'剪接位点活性至少部分取决于与U1小核RNA的碱基互补性。在瞬时共表达测定中，有缺陷的5'剪接位点可以在存在代偿性变化的U1的情况下恢复活性，但是尚不清楚这种突变U1 RNA是否可以在哺乳动物细胞中永久表达。我们已经探索了这个问题，以确定具有改变的特异性的U1小核RNA可能对挽救靶向突变基因或改变前mRNA加工概况是否有价值。这项工作是在我们观察到U1对与替代H-ras外显子相关的剪接位点具有特异性后，在瞬时分析中显着降低潜在致癌性p21ras蛋白的合成后开始的。我们描述了一种哺乳动物互补系统的发展，该系统选择去除放置在耐药基因内的剪接缺陷内含子。观察到与转染的突变U1基因和不同的缺陷剪接位点之间的互补程度成比例的互补，发现以此方式选择的所有细胞均表达突变U1 RNA。另外，这些细胞显示出由未连接的报道基因呈现的缺陷剪接位点的特异性活化。我们讨论这种方法的前景，以永久改变哺乳动物细胞中靶基因的表达。

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