High-affinity hnRNP A1 binding sites and duplex-forming inverted repeats have similar effects on 5′ splice site selection in support of a common looping out and repression mechanism
High-affinity hnRNP A1 binding sites and duplex-forming inverted repeats have similar effects on 5′ splice site selection in support of a common looping out and repression mechanism
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DOI：
10.1017/s1355838202024056
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发表时间：
2002-08-01
期刊：
影响因子：
4.5
通讯作者：
Chabot, B
中科院分区：
文献类型：
--
作者：
Nasim, FUH;Hutchison, S;Chabot, B
hnRNP All蛋白的高亲和力结合位点刺激哺乳动物前mRNA中远端5'剪接位点的使用。值得注意的是，强烈的A1介导的剪接位点选择的转变并不伴随着竞争性5'剪接位点上含有U1 snRNP的复合物的组装的等效变化。为了解释上述结果，我们提出了hnRNP All分子与高亲和力位点结合的相互作用环出了内部5'剪接位点。在这里，我们提出了更多的证据来支持循环模型。首先，用可以通过RNA双链体形成产生环的序列替换所有结合位点以等同的方式激活远端5'剪接位点的使用。第二，增加内部51剪接位点和侧翼A1结合位点之间的距离不会损害远端5'剪接位点的激活。用携带反向重复序列的前体mRNA获得了类似的结果。使用一个前体mRNA只含有一个51剪接位点，我们表明，剪接被抑制时，侧翼由两个高亲和力的A1结合位点或反向重复序列，和内部5'剪接位点的失活是足以引起一个强烈的增加使用的远端供体位点。我们的研究结果与以下观点一致：A1与高亲和力位点的结合促进了环的形成，这一事件将抑制内部5'剪接位点并导致远端5'剪接位点活化。
High-affinity binding sites for the hnRNP All protein stimulate the use of a distal 5' splice site in mammalian pre-mRNAs. Notably, strong A1-mediated shifts in splice site selection are not accompanied by equivalent changes in the assembly of U1 snRNP-containing complexes on competing 5' splice sites. To explain the above results, we have proposed that an interaction between hnRNP All molecules bound to high-affinity sites loops out the internal 5' splice site. Here, we present additional evidence in support of the looping out model. First, replacing All binding sites with sequences that can generate a loop through RNA duplex formation activates distal 5' splice site usage in an equivalent manner. Second, increasing the distance between the internal 51 splice site and flanking A1 binding sites does not compromise activation of the distal 5' splice site. Similar results were obtained with pre-mRNAs carrying inverted repeats. Using a pre-mRNA containing only one 51 splice site, we show that splicing is repressed when flanked by two high-affinity A1 binding sites or by inverted repeats, and that inactivation of the internal 5' splice site is sufficient to elicit a strong increase in the use of the distal donor site. Our results are consistent with the view that the binding of A1 to high-affinity sites promotes loop formation, an event that would repress the internal 5' splice site and lead to distal 5' splice site activation.