Ladd Andrea N, Stenberg Myrna G, Swanson Maurice S, Cooper Thomas A
Department of Pathology, Baylor College of Medicine, Houston, Texas 77030, USA.
Dev Dyn. 2005 Jul;233(3):783-93. doi: 10.1002/dvdy.20382.
Cardiac troponin T (cTNT) exon 5 splicing is developmentally regulated such that it is included in embryonic but not adult heart. CUG-BP and ETR-3-like factor (CELF) proteins promote exon inclusion, whereas polypyrimidine tract binding protein (PTB) and muscleblind-like (MBNL) proteins repress inclusion. In this study, we addressed what happens to these regulatory proteins during heart development to shift the regulatory balance of cTNT alternative splicing. Using dominant-negative proteins, we found that both CELF and PTB activities are required for appropriate splicing in cardiomyocytes. Two CELF proteins, CUG-BP and ETR-3, are nuclear and cytoplasmic in embryonic heart but are down-regulated in adult heart concomitant with loss of exon inclusion. In contrast, PTB and MBNL1 are expressed throughout heart development. The patterns of cTNT splicing and expression of its regulatory factors are conserved between mouse and chicken. Thus, alternative splicing is determined by a balance between positive and negative regulation, and modulation of expression levels of auxiliary splicing regulators may drive developmental splicing changes. ETR-3 and CUG-BP proteins are also down-regulated in other tissues during development, suggesting that CELF proteins play a broad role in developmental splicing regulation.
心肌肌钙蛋白T（cTNT）外显子5的剪接受发育调控，在胚胎心脏中包含该外显子，而在成体心脏中则不包含。CUG结合蛋白和ETR-3样因子（CELF）蛋白促进外显子的包含，而多嘧啶序列结合蛋白（PTB）和类肌肉盲蛋白（MBNL）则抑制外显子的包含。在本研究中，我们探讨了在心脏发育过程中这些调控蛋白发生了什么变化，从而改变了cTNT可变剪接的调控平衡。使用显性负性蛋白，我们发现CELF和PTB的活性对于心肌细胞中的适当剪接都是必需的。两种CELF蛋白，即CUG结合蛋白和ETR-3，在胚胎心脏中存在于细胞核和细胞质中，但在成体心脏中随着外显子包含的缺失而下调。相反，PTB和MBNL1在心脏发育过程中均有表达。cTNT的剪接模式及其调控因子的表达在小鼠和鸡之间是保守的。因此，可变剪接由正负调控之间的平衡决定，辅助剪接调节因子表达水平的调节可能驱动发育过程中的剪接变化。ETR-3和CUG结合蛋白在发育过程中在其他组织中也下调，这表明CELF蛋白在发育剪接调控中发挥广泛作用。
