Investigating Casc3 function in localized mRNA regulation and neuromuscular development

胚胎发育需要对基因表达进行高度特异的时空调控，以建立不同的细胞类型。这种控制在一定程度上是通过调节mRNAs稳定性和表达的RNA结合蛋白实现的。由于RNA调节和神经元定位的动态性质，神经发育对转录后调节中的扰动特别敏感。外显子连接复合体(EJC)通过支持神经前体细胞的增殖而对大脑发育是必需的；此外，EJC核心成分Rbm8a和Magoh对于斑马鱼胚胎发生过程中运动神经元和肌肉的正常发育至关重要。EJC是沉积在剪接的mRNAs上的一种保守的大型多蛋白复合体，在高等真核生物中作为mRNA质量控制的调节因子。EJC可以引起不正确剪接的mRNAs的无义介导的衰变(NMD)，也可以引起含有3‘非翻译区内含子(3UIs)的mRNAs。癌症易感性候选3(Casc3)蛋白是保守的外显子-外显子连接复合体(EJC)的一个亚单位，它似乎加强了转录子子集的衰退。此外，Casc3在信使核糖核酸定位中有几个已知的作用。虽然Casc3基因最初被发现是作为一种因子在乳腺癌患者的转移淋巴结中上调，但Casc3也在神经系统中高度表达，并在斑马鱼的整个发育过程中表达。本研究旨在探讨Casc3在神经肌肉发育过程中调控局部mRNA表达的生物学作用和分子功能。我将利用斑马鱼作为一个模型系统来研究Casc3在神经肌肉发育中的作用。我还将描述Casc3定位的机制，以及它们如何与运动神经元中特定的mRNA特征和命运相关。我假设Casc3是必需的神经肌肉发育，就像活体中的其他EJC核心亚单位一样，并且Casc3促进了含有3UI的mRNAs在运动神经元中的定位和衰退。我们预计Casc3的功能可以延伸到其他表达它的细胞环境中；例如，在神经元环境中剖析Casc3的功能可能有助于深入了解其过度表达如何导致转移癌。此外，这项拟议的研究将在分子水平上加深我们对Casc3的mRNA定位功能的理解，这将广泛地与其在这些生物环境中的功能相关。

Embryonic development requires highly specific spatiotemporal control of gene expression to establish diverse cell types. This control is achieved, in part, by RNA binding proteins that regulate the stability and expression of mRNAs. Due to the dynamic nature of RNA regulation and localization in neurons, neurodevelopment is especially sensitive to perturbations in posttranscriptional regulation. The Exon Junction Complex (EJC) is necessary for brain development through supporting the proliferation of neural progenitors; moreover, EJC core components Rbm8a and Magoh are critical for proper development of motor neurons and muscle during embryogenesis in zebrafish. The EJC is a conserved large multiprotein complex deposited on spliced mRNAs that serves as a regulator of mRNA quality control in higher eukaryotes. The EJC can elicit Nonsense-Mediated Decay (NMD) of improperly spliced mRNAs, but also mRNAs containing 3′ Untranslated Region introns (3UIs). The Cancer Susceptibility Candidate 3 (Casc3) protein is a subunit of the conserved exon-exon junction complex (EJC) that appears to potentiate the decay of a subset of transcripts. Moreover, Casc3 has several documented roles in mRNA localization. While the Casc3 gene was first discovered as a factor that is upregulated in metastatic lymph nodes of breast cancer patients, Casc3 is also highly expressed in the nervous system, and expressed throughout development in zebrafish. This proposal aims to address the biological roles and molecular functions of Casc3 in the control of localized mRNA expression in neuromuscular development. I will utilize zebrafish as a model system to investigate the role of Casc3 in neuromuscular development. I will also characterize Casc3 mechanisms of localization and how they are relevant to specific mRNA features and fates in motor neurons. I hypothesize that Casc3 is necessary neuromuscular development, like other EJC core subunits in vivo, and that Casc3 facilitates localization and decay of 3UI-containing mRNAs in motor neurons. We anticipate that Casc3 function extends to other cellular contexts where it is expressed; for example, dissecting Casc3 functions in a neuronal context may provide insight to how its overexpression could contribute to metastatic cancer. Furthermore, this proposed research will enhance our understanding of the mRNA localization function of Casc3 at the molecular level, which will broadly be relevant to its function in these biological contexts.