The Trinucleotide Repeat Containing 6a-Mediated miRNA Activities in the Ciliogenesis of Airway Epithelium

 描述(由申请人提供)：Tnrc6a蛋白是miRNA介导的基因沉默所必需的，这是有机体正常发育、内稳态和功能所需的基本机制。在体外培养的哺乳动物细胞中，Tnrc6a(GW182)及其相关的胞质颗粒GW/P体(GWB)的功能和机制已经得到了很好的研究。然而，GWBS的形成意义尚不清楚。此外，Tnrc6a和GWBS在体内发育过程中的表达、定位和功能仍然知之甚少。为了在体内研究Tnrc6a和GWBS，我们建立了Tnrc6a突变小鼠，并证明Tnrc6a是miRNA诱导基因沉默所必需的。在这个提案中，我们将扩大我们的研究范围，以了解与Tnrc6a和GWBS相关的亚细胞定位的miRNA机制。我们有值得注意的初步观察，Tnrc6a在多毛细胞中高表达，Tnrc6a蛋白集中在与中心粒/基底体密切相关的GWB中。此外，Tnrc6a的表达中断导致纤毛形成的主要缺陷。在Aim1中，我们建议确定Tnrc6a如何通过影响纤毛发生的关键细胞事件来调节纤毛的形成和功能。在目标2中，我们建议严格鉴定这些独特的多纤毛细胞特异性GWB，并确定Trnc6a/GWBS的形成和定位在纤毛发生中的功能意义。在Aim3中，我们将确定与这些中心粒相关的GWB相关的miRNA程序，该程序调节纤毛发生。这些研究的成功将使我们能够确定纤毛发生中一种新的亚细胞定位的miRNA机制，该机制与Tnrc6a选择性丰富的表达和GWBS的形成和定位有关。与公共卫生相关：miRNA活性的异常表达和功能与包括原发性睫状肌运动障碍(PCD)在内的人类疾病有关。本申请中提出的研究旨在阐明新的和基本的miRNA机制，这些机制对于多纤毛细胞的正确分化至关重要。这些研究的结果应该有助于深入了解miRNAs如何在发育过程中调节正常的纤毛组装，以及这一过程的放松如何导致人类疾病中的纤毛缺陷。

 DESCRIPTION (provided by applicant): Tnrc6a proteins are essential for miRNA-mediated gene silencing, a fundamental mechanism required for proper development, homeostasis, and function of an organism. The function and mechanism of Tnrc6a (GW182) and its associated cytoplasmic granules, GW/P-bodies (GWBs) have been well studied in mammalian cells cultured in vitro. However, the significance for the formation of GWBs is unclear. Moreover, the expression, localization and function of Tnrc6a and GWBs during development in vivo are still poorly understood. To study Tnrc6a and GWBs in vivo, we have generated Tnrc6a mutant mice, and shown that Tnrc6a is required for miRNA-induced gene silencing. In this proposal, we will expand our studies to understand a subcellular localized miRNA mechanism that is associated with Tnrc6a and GWBs in multiciliated cells of airways. We have remarkable preliminary observations that Tnrc6a is highly expressed in multiciliated cells and Tnrc6a proteins are concentrated in GWBs that are closely associated with centrioles/basal bodies. Moreover, disruption of Tnrc6a expression leads to major defects in cilia formation. In Aim1, we proposed to determine how Tnrc6a regulates cilia formation and function by affecting key cellular events of ciliogenesis. In Aim 2, we proposed to rigorously characterize these unique multiciliated cell-specific GWBs, and determine the functional significance of formation and localization of Trnc6a/GWBs in ciliogenesis. In Aim3, we will identify the miRNA program associated with these centriole associated GWBs that regulates ciliogenesis. Successful accomplishment of these studies will allow us to identify a novel subcellular localized miRNA mechanism in ciliogenesis that is associated with selectively enriched expression of Tnrc6a and the formation and localization of GWBs. RELEVANCE TO PUBLIC HEALTH: Aberrant expression and function of miRNA activities have been associated with human disorders including primary ciliary dyskinesia (PCD). Studies proposed in this application are aimed to elucidate novel and fundamental miRNA mechanisms that are essential for proper differentiation of multiciliated cells. Results from these studies should provide insight into how miRNAs regulate normal cilia assembly during development and how deregulation of this process leads to defective cilia in human disorders.