COOPERATION OF PRE-MESSENGER-RNA SEQUENCE ELEMENTS IN SPLICE SITE SELECTION
COOPERATION OF PRE-MESSENGER-RNA SEQUENCE ELEMENTS IN SPLICE SITE SELECTION
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DOI：
10.1128/mcb.12.5.2108
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发表时间：
1992-05-01
影响因子：
5.3
通讯作者：
KOLE, R
中科院分区：
文献类型：
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作者：
DOMINSKI, Z;KOLE, R
我们最近已经证明，短的内部外显子在前体mRNA转录与三个外显子和两个内含子被忽略的剪接机制在体外和体内，导致外显子跳跃。当上游内含子中多聚嘧啶段的嘧啶含量增加时，外显子跳跃被逆转（Z. Dominski和R.科莱Cell. 11：6075-6083，1991）。在这里，我们表明，跳跃的短的内部外显子可以部分逆转的突变，修改上游分支点序列或5'剪接位点在外显子的末端，其各自的共识序列。当修饰的元件在相同的前体mRNA中彼此组合时，外显子跳跃被完全逆转。当这些元件单独与上游多聚嘧啶道结合时，也观察到外显子跳跃的完全逆转，所述上游多聚嘧啶道由三个嘌呤到嘧啶突变加强。所观察到的剪接位点选择的模式在体外（在HeLa细胞的核提取物中）和体内（在转染的HeLa细胞中）是相似的。我们还表明，下游内含子的长度在剪接位点选择中起着重要作用。我们的数据表明，前体mRNA中序列元件之间的相互作用控制着每个剪接事件的结果，为选择性剪接的非常微妙的调节提供了手段。
We have recently demonstrated that short internal exons in pre-mRNA transcripts with three exons and two introns are ignored by splicing machinery in vitro and in vivo, resulting in exon skipping. Exon skipping is reversed when the pyrimidine content of the polypyrimidine tract in the upstream intron is increased (Z. Dominski and R. Kole, Mol. Cell. Biol. 11:6075-6083, 1991). Here we show that skipping of the short internal exon can be partially reversed by mutations which modify the upstream branch point sequence or the 5' splice site at the end of the exon to their respective consensus sequences. When the modified elements are combined with one another in the same pre-mRNA, exon skipping is fully reversed. Full reversion of exon skipping is also observed when these elements are combined individually with the upstream polypyrimidine tract strengthened by three purine-to-pyrimidine mutations. The observed patterns of splice site selection are similar in vitro (in nuclear extracts from HeLa cells) and in vivo (in transfected HeLa cells). We also show that the length of the downstream intron plays a role in splice site selection. Our data indicate that the interplay between the sequence elements in pre-mRNA controls the outcome of each splicing event, providing the means for very subtle regulation of alternative splicing.