SKIP蛋白复合体的组装及在Notch信号介导的肌肉发生中的作用

Notch signaling has a simple framework that is evolutionarily conserved in all metazoans. Despite the simple design of the core pathway, Notch signaling affects the cell fate decisions not only across a broad spectrum of animal kingdom, but also across a wide range of different cell types in one organism and at multiple steps during cell lineage commitment. Emerging evidence has pointed to this pathway as a key regulator in embryonic muscle development, adult muscle homeostasis and regeneration. However, the molecular mechanisms underlying the specificity of the temporal and spatial regulation of myogenesis by Notch signaling remain largely unknown. Notch signaling is initiated by extracellular interactions between Notch receptors and ligands. Following a serial of proteolytic cleavage of the receptor, the freed intracellular domain of Notch (NICD) translocates from the cytoplasm to the nucleus and forms transcriptionally active complex with its coactivators. The nuclear coregulators of NICD are critical for the fine-tuning activation of Notch target genes. SKIP is both an integral component of active spliceosome and a transcription elongation regulator for induced genes. SKIP has been reported to function as a nuclear cofactor of Notch and is required for Notch signaling activation. Our preliminary data showed that SKIP is highly expressed in embryonic muscle progenitor cells and mainly locates at the nuclear periphery. We also found that SKIP undergoes significant posttranslational modification during the C2C12 myoblast cell differentiation. Through proteomics analysis, we identified SKIP interacting proteins. We found that the Homer family proteins strongly associate with SKIP both in vitro and in vivo. Biochemical and functional studies indicate that SKIP exists in two functionally antagonistic protein complexes. Collectively, these data suggest that the dynamic balance between these two complexes may control SKIP transcriptional and splicing activity and its function in myogenesis. The central objective of this project is to elucidate the molecular mechanisms by which the SKIP protein complexes assemble and regulate Notch signaling-mediated myogenesis. To accomplish this goal, we will address the following key questions: (1) How do SKIP protein complexes assemble in vitro and in vivo? (2) What are the effects of Notch signaling alteration and myoblast cell differentiation on the dynamic balance between these two complexes? (3) What roles do SKIP protein complexes play in Notch-regulated myogenesis? (4) How do SKIP protein complexes regulate the Notch target gene expression? These studies will not only illuminate the molecular details that underlie the assembly and biological function of SKIP protein complexes, but also will greatly improve our understanding of how Notch siganling specifically operates in different temporal and spatial cellular contexts.

Notch信号在机体发育过程中发挥重要作用，但其组织特异性的调控机制仍有待进一步阐明。在Notch信号激活下游靶基因表达的过程中，细胞核内的协同分子起重要的调控作用。转录延伸和RNA剪接分子SKIP是Notch信号的胞核内的重要协同调节蛋白。我们前期预实验的结果表明SKIP特异性地在小鼠胚胎的成肌前体细胞中高表达，提示其可能组织特异性地调控Notch信号介导的肌肉发生。通过蛋白质组学研究，我们发现了SKIP的新的相互作用蛋白，初步实验结果提示SKIP存在于两个功能相互拮抗的蛋白复合体中。拟进行的工作将研究SKIP蛋白复合体的组装机制、对Notch信号通路下游靶基因的调控及其在Notch信号介导的肌肉发生过程中的作用。此研究不仅将有助于阐释SKIP蛋白复合体的生物学功能、在转录延伸及其偶联的RNA剪接过程中的调控作用，还能够帮助我们理解Notch信号通路调节肌肉发生的时空特异性的分子机制。

Notch信号在肌肉发生过程中发挥重要的作用，但其组织特异性的调控机制仍有待阐明。Notch信号的活化及对下游靶基因表达的调控需要细胞核内的协同调控分子。转录延伸和RNA剪接分子SKIP是Notch信号的胞核内的重要协同调节蛋白。本项目通过生物化学与分子生物学手段，研究SKIP蛋白复合体体内体外的组装机制，探讨其调控转录延伸的分子机制，并研究了SKIP蛋白的生理功能。我们发现：（1） SKIP存在于两个不同的蛋白复合体，即SKIP/PRP19复合体和SKIP/PPIL1/Homer复合体。（2） SKIP是Notch的胞核内的重要协同调控分子。（3） SKIP特异地在胚胎成肌前体细胞中高表达，在肌肉前体细胞中敲低SKIP的基因表达抑制肌肉的分化。后期进一步研究发现，SKIP蛋白在体内与体外具有形成相分离的能力，SKIP蛋白参与调控pre-mRNA剪接和姐妹染色单体粘连的相分离相关机制。这一工作不仅将有助于阐释SKIP蛋白复合体的生物学功能、在转录延伸及其偶联的RNA剪接过程中的调控作用，还能够帮助我们理解Notch信号通路调节肌肉发生的时空特异性的分子机制。.除此之外，本项目组同时还阐释了SKIP相互作用蛋白P-TEFb激酶在多囊肾病发生发展中的作用。我们发现：（1）多囊肾病小鼠模型中P-TEFb激酶复合体蛋白表达增加、底物磷酸化水平升高。（2）抑制P-TEFb激酶活性延缓多囊肾病囊肿发展。