Centre de Génétique Moléculaire, FRE 3144, CNRS, Gif sur Yvette, France, Université Paris XI, Orsay, France.
Exp Cell Res. 2011 Jan 1;317(1):94-106. doi: 10.1016/j.yexcr.2010.09.010. Epub 2010 Sep 25.
Exon 6B from the chicken β-tropomyosin pre-mRNA is alternatively spliced during myogenic differentiation. Exon 6B is excluded in mRNA from myoblasts and included in mRNA from myotubes. We investigated the regulation of exon 6B inclusion ex vivo in a quail myogenic cell line, which behaves as myoblasts in undifferentiated state and as myotubes after differentiation. We show that the β-tropomyosin exon 6B is a novel target of CUG-BP and ETR-3-like factor (CELF). Overexpression of CELF proteins in myoblasts activates splicing of exon 6B. Using a dominant-negative form of CELF4, we demonstrate that CELF proteins are involved in switching splicing from exon 6A towards exon 6B inclusion during myogenic differentiation. We also found that polypyrimidine tract binding protein (PTB) is required for splicing repression of exon 6B in myoblasts. CELF and PTB proteins exhibit antagonistic properties toward inclusion of exon 6B during myogenic differentiation. Our results suggest that a change in the protein level of CUGBP1 and PTB proteins, associated with a distinct pattern of PTB during the transition from myoblasts to myotubes is one of the parameters involved in regulating splicing of exon 6B during myogenesis.
鸡β-原肌球蛋白前体 mRNA 的外显子 6B 在肌发生分化过程中发生可变剪接。外显子 6B 在成肌细胞的 mRNA 中被排除，而在肌管的 mRNA 中被包含。我们在鹌鹑肌细胞系中进行了体外研究，该细胞系在未分化状态下表现为成肌细胞，分化后表现为肌管。我们表明β-原肌球蛋白外显子 6B 是 CUG-BP 和 ETR-3 样因子（CELF）的新靶标。在成肌细胞中过表达 CELF 蛋白可激活外显子 6B 的剪接。使用 CELF4 的显性负形式，我们证明 CELF 蛋白参与肌发生分化过程中外显子 6A 向 6B 内含子剪接的转换。我们还发现多嘧啶 tract 结合蛋白（PTB）是成肌细胞中外显子 6B 剪接抑制所必需的。CELF 和 PTB 蛋白在肌发生分化过程中外显子 6B 的内含子中表现出拮抗性质。我们的结果表明，CUGBP1 和 PTB 蛋白的蛋白水平变化，以及 PTB 在成肌细胞向肌管的转变过程中的独特模式，是调节外显子 6B 剪接的一个参数。
