Local translation and viral infection in the airway epithelium

项目摘要-摘要 多纤毛细胞（MCCs）是气道上皮的关键组成部分，在粘膜纤毛功能中起着重要作用。 清除，肺部抵御吸入病原体的第一道防线。MCCs是众所周知的病毒靶点， 病原体，包括甲型流感、SARS-COVID 2和呼吸道合胞病毒（RSV），通常会导致严重的 呼吸系统疾病，有长期后遗症和发病率。但是，在这方面仍然存在着重大的知识空白。 这些病原体引发气道疾病的机制。MCC需要生产和顶端定位 在多纤毛发生过程中，每个细胞数百个纤毛的组装所必需的大量蛋白质。的 允许这些蛋白质在MCC中有效产生和局部翻译的机制仍然很差 明白有证据表明，在病毒感染期间，宿主的细胞翻译机制被劫持， 产生病毒蛋白进行复制。如何在MCC中建立局部翻译并在病毒感染中靶向 将在本提案中进行研究。我们的初步研究揭示了翻译起始的显著表现 与miRNAs共定位的核糖体蛋白和新生多肽，三核苷酸重复序列， 含有6a（TNRC 6a）和Argonaute 2（AGO 2）的未成熟MCC经历多纤毛发生。这些 信号集中在尚未报道的顶端细胞质颗粒中，我们将其命名为局部 翻译颗粒（LT颗粒）。令人惊讶的是，与传统上与miRNA相关的其他颗粒不同， 功能，没有酶所需的mRNA降解检测LT颗粒。相反，LT颗粒是 蛋白质翻译的高活性位点。值得注意的是，干扰Tnrc 6a表达，干扰局部翻译和 导致缺陷性多纤毛形成，我们也在RSV感染的MCC中观察到这种表型。在这里我们将测试 假设i）miRNA途径的组分募集mRNA的亚群，并且 机器到LT颗粒，用于MCC分化期间局部有效的大规模蛋白质合成; ii）破坏 这种局部翻译程序的启动是RSV感染发病机制中的关键决定因素。因此，我们建议 确定靶向mRNA（Aim 1）和招募翻译机器（Aim 2）到LT颗粒的机制， MCCs，并确定RSV如何破坏这些机制感染人气道上皮细胞（目的3）。的 从这些研究中产生的知识将大大促进我们对当地机制的理解。 多纤毛发生中的翻译和呼吸道RSV感染的发病机制。 与公共卫生的相关性：多纤毛细胞（MCC）是防御的关键组成部分 肺的机制。在未成熟的上皮细胞中的病毒感染可以导致破坏性的呼吸道疾病。 对呼吸功能受损的婴儿或成人的影响。这些研究产生的新信息 将促进我们对纤毛发生如何调节以及病毒感染（如RSV）如何调节纤毛发生的理解。 破坏未成熟呼吸道纤毛的形成。

Project Summary - Abstract Multiciliated cells (MCCs) are key components of the airway epithelium playing a major role in mucociliary clearance, the first line of lung defense against inhaled pathogens. MCCs are well-known targets of viral pathogens, including influenza A, SARS-COVID2 and respiratory syncytial virus (RSV), often resulting in severe respiratory conditions with long-term sequelae and morbidity. Still there are major gaps of knowledge on the mechanisms by which these pathogens trigger airway disease. MCCs require production and apical localization of a large number of proteins essential for the assembly of hundreds of cilia per cell during multiciliogenesis. The mechanisms that allow efficient production and local translation of these proteins in MCCs are still poorly understood. There is evidence that during viral infection, the host’s cellular translation machinery is hijacked to produce viral proteins for replication. How local translation is established in MCCs and targeted in viral infection will be studied in this proposal. Our preliminary studies revealed a striking expression of translation initiation factors (eIFs), ribosomal proteins and nascent polypeptides colocalized with miRNAs, Trinucleotide repeat- containing 6a (TNRC6a) and Argonaute 2 (AGO2) in immature MCCs undergoing multiciliogenesis. These signals were concentrated in not yet reported apical cytoplasmic granules, which we named as Localized Translation granules (LT granules). Surprisingly, unlike other granules traditionally associated with miRNA function, no enzymes required for mRNA degradation were detected in LT granules. Instead, LT granules were highly active sites of protein translation. Notably, disrupting Tnrc6a expression, disturbed local translation and resulted in defective multicilia formation, a phenotype we also observed in RSV-infected MCCs. Here we will test the hypotheses that i) components of the miRNA pathway recruit subsets of mRNAs and the translation machinery to LT granules for local efficient large-scale protein synthesis during MCC differentiation; ii) disruption of this local translation program is a key determinant in the pathogenesis of RSV infection. Thus, we propose to identify mechanisms that target mRNAs (Aim 1) and recruit the translation machinery (Aim 2) to LT granules in MCCs, and determine how RSV disrupts these mechanisms to infect the human airway epithelium (Aim 3). The knowledge generated from these studies will significantly advance our understanding of the mechanisms of local translation in multiciliogenesis and the pathogenesis of RSV infection in the respiratory tract. RELEVANCE TO PUBLIC HEALTH: Multiciliated cells (MCCs) are crucial components of the defense mechanisms of the lung. Viral infections in the immature epithelium of conducting airways can have devastating effects in infants or adults with compromised respiratory function. Novel information generated from these studies will advance our understanding of how proper ciliogenesis is regulated, and how viral infections, such as RSV disrupt cilia formation in immature respiratory tract.