REGULATION OF ALTERNATIVE SPLICING IN MUSCLE BY ETR-3

这个项目的目标是了解横纹肌发育过程中调节选择性剪接的机制。心肌肌钙蛋白T（cTNT）在横纹肌中经历发育调节的选择性切割，并且是细胞特异性剪接调节的有用模型。cTNT前体mRNA含有肌肉特异性剪接增强子（MSE），这是在胚胎横纹肌中观察到的增强的选择性外显子包含所需的内含子元件。ETR-3是RNA结合蛋白保守家族的成员，结合MSE并促进MSE依赖性外显子包含。ETR-3在骨骼肌分化过程中被诱导，并在心脏发育过程中经历同种型转变。这两种变化都与cTNT剪接中的调节开关相关。这些结果强烈表明，ETR-3是横纹肌发育中选择性剪接的主要调节因子。为了确定ETR-3在肌肉特异性选择性剪接中的作用，我将：（i）将ETR-3表征为骨骼肌分化过程中选择性剪接的正调控因子，（ii）确定心脏发育过程中ETR-3亚型转换的意义，（iii）确定ETR-3在横纹肌发育过程中协调调节选择性剪接的作用。ETR-3与CUG-BP具有78%的同一性，CUG-BP被认为介导强直性肌营养不良的发病机制，强直性肌营养不良是一种由DMPK基因的3' UTR中不稳定CTG重复序列的扩增引起的常染色体显性遗传疾病。扩增的DMPK转录物在细胞核中积累，并且被认为通过破坏CUG-BP的功能来影响其他基因的转录后加工。我们的研究结果表明，ETR-3是一个同样可能的候选人调解反式显性效应在DM。

The goal of this project is to understand the mechanisms that regulate alternative splicing during striated muscle development. Cardiac troponin T (cTNT) undergoes developmentally regulated alternative slicing in striated muscle, and is a useful model for cell-specific splicing regulation. cTNT pre-mRNAs contain muscle-specific splicing enhancers (MSEs), intronic elements that are required for the enhanced alternative exon inclusion that is observed in embryonic striated muscle. ETR-3, a member of a conserved family of RNA-binding proteins, binds MSEs and promotes MSE-dependent exon inclusion. ETR-3 is induced during skeletal muscle differentiation and undergoes an isoform transition during heart development. Both of these changes correlate with regulated switches in cTNT splicing. These results strongly suggest that ETR-3 is a major regulator of alternative splicing in striated muscle development. To determine the role of ETR-3 in muscle-specific alternative splicing, I will: (i) Characterize ETR-3 as a positive regulator of alternative splicing during skeletal muscle differentiation, (ii) Determine the significance of the ETR-3 isoform transition during heart development, and (iii) Determine the role of ETR-3 in coordinated regulation of alternative splicing during striated muscle development. ETR-3 is 78% identical to CUG-BP, which is proposed to mediate pathogenesis of myotonic dystrophy, an autosomal dominant genetic disorder caused by an expansion of unstable CTG repeats in the 3' UTR of the DMPK gene. Expanded DMPK transcripts accumulate in the nucleus, and are thought to affect post-transcriptional processing of other genes by disrupting function of CUG-BP. Our results suggest that ETR-3 is an equally likely candidate for mediating a trans-dominant effect in DM.