Bonano Vivian I, Oltean Sebastian, Brazas Robert M, Garcia-Blanco Mariano A
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, 27710, USA.
RNA. 2006 Dec;12(12):2073-9. doi: 10.1261/rna.248506. Epub 2006 Oct 26.
Alternative splicing multiplies genomic coding capacity and regulates proteomic composition. A well-studied example of this plasticity leads to the synthesis of functionally distinct isoforms of the Fibroblast Growth Factor Receptor-2 (FGFR2). The regulation of this isoform diversity necessitates the silencing of FGFR2 exon IIIb, which is mediated by flanking intronic splicing silencers and the polypyrimidine tract binding protein (PTB). To visualize this splicing decision in vivo, we developed mice harboring a green fluorescent protein construct that reports on the silencing of exon IIIb. The animals also harbor a red fluorescent protein reporter of constitutive splicing as an allelic control. This dual reporter system revealed that in various organs and cell types the silencing of exon IIIb required the intronic silencers. In neurons, which do not express PTB, we observed robust silencer-dependent repression of exon IIIb, suggesting that the neural paralog, brain PTB, can take over this function. In the epidermis, however, the intronic silencers were not required for efficient silencing. This work provides a first glimpse at splicing regulation among different cell types in vivo and promises the drafting of an anatomic map of splicing decisions.
可变剪接增加了基因组的编码能力并调节蛋白质组的组成。一个对此可塑性进行了充分研究的例子是成纤维细胞生长因子受体2（FGFR2）功能不同的异构体的合成。这种异构体多样性的调节需要沉默FGFR2外显子IIIb，这是由侧翼内含子剪接沉默子和多聚嘧啶序列结合蛋白（PTB）介导的。为了在体内可视化这种剪接决定，我们构建了携带绿色荧光蛋白构建体的小鼠，该构建体报告外显子IIIb的沉默情况。这些动物还携带一个组成型剪接的红色荧光蛋白报告基因作为等位基因对照。这个双报告系统表明，在各种器官和细胞类型中，外显子IIIb的沉默需要内含子沉默子。在不表达PTB的神经元中，我们观察到外显子IIIb有强大的沉默子依赖性抑制，这表明神经旁系同源物脑PTB可以接管这个功能。然而，在表皮中，高效沉默不需要内含子沉默子。这项工作首次展示了体内不同细胞类型之间的剪接调控，并有望绘制出一张剪接决定的解剖图谱。
