Competing Upstream 5′ Splice Sites Enhance the Rate of Proximal Splicing
Competing Upstream 5′ Splice Sites Enhance the Rate of Proximal Splicing
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DOI：
10.1128/mcb.01071-09
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发表时间：
2010-04-15
影响因子：
5.3
通讯作者：
Hertel, Klemens J.
中科院分区：
文献类型：
--
作者：
Hicks, Martin J.;Mueller, William F.;Hertel, Klemens J.
选择性5'剪接位点选择是导致mRNA多样化的主要途径之一。这种类型的选择性剪接的调节取决于激活或抑制竞争剪接位点的使用的调节元件的存在，通常导致优先使用近端剪接位点。然而，参与近端剪接位点选择的机制和近端剪接位点实现的热力学优势还没有得到很好的理解。在这里，我们进行了系统的分析，选择5'剪接位点的使用，在体外剪接测定。我们发现，观察到的剪接率与它们的U1 snRNA碱基配对潜力密切相关。弱的U1 snRNA与5'剪接位点的相互作用被下游外显子的邻近显著拯救，表明剪接位点识别的内含子定义模式是高效的。在竞争剪接位点的情况下，与下游3'剪接位点的接近度在决定剪接位点选择方面比实际5'剪接位点/U1 snRNA碱基配对潜力更有影响力。令人惊讶的是，动力学分析还表明，上游竞争性5'剪接位点提高了近端剪接的速率。这些结果揭示了一个新的剪接调控元件的发现，上游5'剪接位点作为剪接增强子发挥作用。
Alternative 5' splice site selection is one of the major pathways resulting in mRNA diversification. Regulation of this type of alternative splicing depends on the presence of regulatory elements that activate or repress the use of competing splice sites, usually leading to the preferential use of the proximal splice site. However, the mechanisms involved in proximal splice site selection and the thermodynamic advantage realized by proximal splice sites are not well understood. Here, we have carried out a systematic analysis of alternative 5' splice site usage using in vitro splicing assays. We show that observed rates of splicing correlate well with their U1 snRNA base pairing potential. Weak U1 snRNA interactions with the 5' splice site were significantly rescued by the proximity of the downstream exon, demonstrating that the intron definition mode of splice site recognition is highly efficient. In the context of competing splice sites, the proximity to the downstream 3' splice site was more influential in dictating splice site selection than the actual 5' splice site/U1 snRNA base pairing potential. Surprisingly, the kinetic analysis also demonstrated that an upstream competing 5' splice site enhances the rate of proximal splicing. These results reveal the discovery of a new splicing regulatory element, an upstream 5' splice site functioning as a splicing enhancer.