RNA folding affects the recruitment of SR proteins by mouse and human polypurinic enhancer elements in the fibronectin EDA dxon
RNA folding affects the recruitment of SR proteins by mouse and human polypurinic enhancer elements in the fibronectin EDA dxon
复制标题
DOI：
10.1128/mcb.24.3.1387-1400.2004
复制
发表时间：
2004-02-01
影响因子：
5.3
通讯作者：
Baralle, FE
中科院分区：
文献类型：
--
作者：
Buratti, E;Muro, AF;Baralle, FE
在人类中，纤维连接蛋白EDA外显子的包含或排除主要由多尿酸增强元件（外显子剪接增强子[ESE]）和附近的沉默元件（外显子剪接沉默元件[ESS]）调节。虽然人类和小鼠的ESS表现相同，但引入同源小鼠ESS序列的突变要么不会改变剪接效率，要么会完全排除外显子。在这里，我们表明这种明显矛盾的行为不能简单地用两个物种之间的局部序列差异来解释。相反，核苷酸的差异作为一个整体决定了各自RNA二级结构的一些变化。通过比较两种不同的结构对其假定的ESS序列同源缺失的反应，我们发现剪接行为的变化可以通过两种rna中不同的ESE显示来解释。SR蛋白与每个外显子结合水平的rna -蛋白相互作用分析证实了这一点。免疫沉淀模式显示存在复杂的多sr蛋白- rna相互作用，在引入ESE和ESS变异后，这些相互作用随着二级结构的变化而消失。综上所述，我们的研究结果表明，除了一级序列的同一性外，序列背景对能够影响pre-mRNA剪接的功能单元的形成也有很大的影响。
In humans, inclusion or exclusion of the fibronectin EDA exon is mainly regulated by a polypurinic enhancer element (exonic splicing enhancer [ESE]) and a nearby silencer element (exonic splicing silencer [ESS]). While human and mouse ESEs behave identically, mutations introduced into the homologous mouse ESS sequence result either in no change in splicing efficiency or in complete exclusion of the exon. Here, we show that this apparently contradictory behavior cannot be simply accounted for by a localized sequence variation between the two species. Rather, the nucleotide differences as a whole determine several changes in the respective RNA secondary structures. By comparing how the two different structures respond to homologous deletions in their putative ESS sequences, we show that changes in splicing behavior can be accounted for by a differential ESE display in the two RNAs. This is confirmed by RNA-protein interaction analysis of levels of SR protein binding to each exon. The immunoprecipitation patterns show the presence of complex multi-SR protein-RNA interactions that are lost with secondary-structure variations after the introduction of ESE and ESS variations. Taken together, our results demonstrate that the sequence context, in addition to the primary sequence identity, can heavily contribute to the making of functional units capable of influencing pre-mRNA splicing.