Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697-4025, USA.
Mol Cell Biol. 2010 Apr;30(8):1878-86. doi: 10.1128/MCB.01071-09. Epub 2010 Feb 1.
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.
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，近端剪接位点选择的机制以及近端剪接位点实现的热力学优势还不是很清楚。在这里，我们使用体外剪接实验对选择性 5' 剪接位点的使用进行了系统分析。我们表明，观察到的剪接率与它们与 U1 snRNA 的碱基配对潜力密切相关。弱 U1 snRNA 与 5' 剪接位点的相互作用通过下游外显子的接近得到了显著挽救，证明了剪接位点识别的内含子定义模式非常有效。在竞争剪接位点的情况下，下游 3' 剪接位点的接近对剪接位点选择的影响大于实际的 5' 剪接位点/U1 snRNA 碱基配对潜力。令人惊讶的是，动力学分析还表明，上游竞争 5' 剪接位点增强了近端剪接的速度。这些结果揭示了一种新的剪接调节元件的发现，即上游 5' 剪接位点作为剪接增强子发挥作用。
