SUPPRESSION OF MAMMALIAN 5' SPLICE-SITE DEFECTS BY U1 SMALL NUCLEAR RNAS FROM A DISTANCE
SUPPRESSION OF MAMMALIAN 5' SPLICE-SITE DEFECTS BY U1 SMALL NUCLEAR RNAS FROM A DISTANCE
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DOI：
10.1073/pnas.91.22.10470
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发表时间：
1994-10-25
影响因子：
11.1
通讯作者：
LEVINSON, AD
中科院分区：
文献类型：
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作者：
COHEN, JB;SNOW, JE;LEVINSON, AD
在从mRNA前体去除内含子的过程中，最早的事件之一是在U1小核（sn）RNA和5'剪接位点之间建立碱基配对相互作用。在5'剪接位点的突变，阻止剪接，通常可以抑制U1 snRNA的共表达与补偿性变化，但在酵母中，准确的剪接时，普遍保守的第一内含子碱基被改变，不能恢复。在我们的哺乳动物系统中，这样的突变也不能被抑制，但互补的U1引起下游12个碱基的异常剪接。这一结果让人想起在酵母中观察到的异常5'剪接位点可以被U1 snRNA从远处激活。使用快速，定性的蛋白质表达测定，我们提供的证据表明，5'剪接位点突变可以抑制在哺乳动物细胞中的U1 snRNAs与一系列序列的上游或下游的网站。我们的方法在体内将哺乳动物剪接的承诺-激活步骤与5'剪接位点定义的过程分开，因此将有助于两者的遗传表征。
One of the earliest events in the process of intron removal from mRNA precursors is the establishment of a base-pairing interaction between U1 small nuclear (sn) RNA and the 5' splice site. Mutations at the 5' splice site that prevent splicing can often be suppressed by coexpression of U1 snRNAs with compensatory changes, but in yeast, accurate splicing is not restored when the universally conserved first intron base is changed. In our mammalian system as well, such a mutation could not be suppressed, but the complementary U1 caused aberrant splicing 12 bases downstream. This result is reminiscent of observations in yeast that aberrant 5' splice sites can be activated by U1 snRNA from a distance. Using a rapid, qualitative protein expression assay, we provide evidence that 5' splice-site mutations can be suppressed in mammalian cells by U1 snRNAs with complementarity to a range of sequences upstream or downstream of the site. Our approach uncouples in vivo the commitment-activation step of mammalian splicing from the process of 5' splice-site definition and as such will facilitate the genetic characterization of both.