Le Sommer Caroline, Lesimple Michelle, Mereau Agnès, Menoret Severine, Allo Marie-Rose, Hardy Serge
UMR 6061 CNRS-Université de Rennes 1, IFR 140 Faculté de Médecine, CS 34317, 35043 Rennes Cedex, France.
Mol Cell Biol. 2005 Nov;25(21):9595-607. doi: 10.1128/MCB.25.21.9595-9607.2005.
The polypyrimidine tract binding protein (PTB) has been described as a global repressor of regulated exons. To investigate PTB functions in a physiological context, we used a combination of morpholino-mediated knockdown and transgenic overexpression strategies in Xenopus laevis embryos. We show that embryonic endoderm and skin deficient in PTB displayed a switch of the alpha-tropomyosin pre-mRNA 3' end processing to the somite-specific pattern that results from the utilization of an upstream 3'-terminal exon designed exon 9A9'. Conversely, somitic targeted overexpression of PTB resulted in the repression of the somite-specific exon 9A9' and a switch towards the nonmuscle pattern. These results validate PTB as a key physiological regulator of the 3' end processing of the alpha-tropomyosin pre-mRNA. Moreover, using a minigene strategy in the Xenopus oocyte, we show that in addition to repressing the splicing of exon 9A9', PTB regulates the cleavage/polyadenylation of this 3'-terminal exon.
多嘧啶序列结合蛋白（PTB）被认为是可变剪接外显子的全局阻遏物。为了研究PTB在生理环境中的功能，我们在非洲爪蟾胚胎中结合使用了吗啉代介导的敲低和转基因过表达策略。我们发现，缺乏PTB的胚胎内胚层和皮肤显示α-原肌球蛋白前体mRNA 3'末端加工转换为体节特异性模式，这是由于使用了上游3'-末端外显子（设计为外显子9A9'）导致的。相反，PTB在体节中的靶向过表达导致体节特异性外显子9A9'的抑制，并转换为非肌肉模式。这些结果证实PTB是α-原肌球蛋白前体mRNA 3'末端加工的关键生理调节因子。此外，我们在非洲爪蟾卵母细胞中使用小基因策略，发现除了抑制外显子9A9'的剪接外，PTB还调节该3'-末端外显子的切割/聚腺苷酸化。
