Regulation and biological functions of mRNA Alternative Polyadenylation in the Brain

项目摘要 选择性多聚腺苷酸化（阿帕）在大脑中产生高度多样的转录组和蛋白质组， 保持正常的神经功能。我们和其他人最近的作品揭示了一种复杂而动态的 APA在大脑中的景观具有许多细胞类型特异性阿帕调节和阿帕在以下方面的新兴作用： 神经系统疾病然而，神经系统疾病中的阿帕改变仍然在很大程度上未被探索， 在脑部疾病中严格设计的体内高通量阿帕研究尤其缺乏， 技术困难。我们在2017年通过开发cTag-PAPERCLIP技术打破了技术壁垒， 它同时描绘了来自完整小鼠大脑的单个细胞类型中的20，000多聚腺苷酸位点， 并且比其他需要细胞纯化的测序策略更好地保留体内转录组。 脑硬化症（TSC）的特征是严重脑梗死患者脑中mTORC 1信号的激活。 神经系统症状有趣的是，mTORC 1激活最近被证明可以引起全球近端 阿帕改变是通过调节非脑细胞中的阿帕因子来实现的，这表明异常的阿帕是一种神经细胞因子。 先前未知的TSC细胞表型。在这项申请中，我们建议进行一项证明- 在本研究中，我们应用cTag-PAPERCLIP通过整合新的工具来研究TSC中的阿帕 我们最近开发的。我们将确定阿帕亚型表达的变化，以响应急性丧失， Tsc 1和通过3种脑细胞类型中的体内cTag-PAPERCLIP谱分析的mTORC 1信号传导的激活： 兴奋性神经元、抑制性神经元和星形胶质细胞。我们将利用我们建立的生物信息学管道， 鉴定每种细胞类型中稳健的和生物学上重要的阿帕变化。我们将进行RNA测序， 为观察到的阿帕变化提供独立验证。阿帕可以改变细胞信号网络 通过产生截短的蛋白质或RNA结合蛋白的3′ UTR调控缺失， microRNAs。对于阿帕变化预测进行这样的影响，我们将实验验证预测 普通细胞系和原代神经元培养物中蛋白质的定量或定性变化。最后，我们将 我们还研究了mTORC 1激活引起阿帕变化的分子机制， 原代神经元培养在拟议的项目完成后，我们希望首次揭示如何 TSC中mTORC 1的激活改变了兴奋性神经元、抑制性神经元和 星形胶质细胞除了解决一个关键问题-阿帕失调如何有助于TSC 发病机制我们的研究结果还将为以下知识和临床管理提供新的见解： TSC和其他具有mTOR通路失调的神经系统疾病（“mTOR病”）

Project Summary Alternative polyadenylation (APA) generates highly diverse transcriptome and proteome in the brain to maintain proper neurological functions. Recent works from others and us revealed a complex and dynamic APA landscape in the brain with many cell type-specific APA regulations and an emerging role of APA in neurological disease. However, APA alterations in neurological disorders remain largely unexplored and rigorously designed in vivo high-throughput APA studies in brain disease are especially lacking due to technical difficulties. We broke the technical barrier in 2017 by developing the cTag-PAPERCLIP technology, which simultaneously profiles more than 20,000 poly(A) sites in individual cell types from intact mouse brain and preserves the in vivo transcriptome better than other sequencing strategies that require cell purification. Tuberous sclerosis (TSC) is characterized by activation of the mTORC1 signaling in the brain with severe neurological symptoms. Interestingly, mTORC1 activation was recently shown to cause global proximal APA shift through regulating an APA factor in non-brain cells, which suggests that aberrant APA is a previously unknown cellular phenotype of TSC. In this application, we propose to conduct a proof-of- principle study in which we apply cTag-PAPERCLIP to investigate APA in TSC by incorporating new tools that we recently developed. We will identify changes in APA isoform expression in response to acute loss of Tsc1 and activation of mTORC1 signaling by in vivo cTag-PAPERCLIP profiling in 3 brain cell types: excitatory neurons, inhibitory neurons and astrocytes. We will use our established bioinformatics pipeline to identify robust and biologically important APA changes in each cell type. We will perform RNA-seq to provide independent validations for the observed APA changes. APA can alter the cellular signaling network through production of truncated proteins or loss of 3′ UTR regulation from RNA-binding proteins and microRNAs. For APA changes predicted to carry such effects, we will experimentally validate the predicted quantitative or qualitative protein changes in common cell lines and primary neuron cultures. Lastly, we will also investigate the molecular mechanisms responsible for APA changes from mTORC1 activation in primary neuron cultures. Upon completion of the proposed project, we expect to reveal for the first time how mTORC1 activation in TSC alters the cellular APA landscapes in excitatory neurons, inhibitory neurons and astrocytes in addition to addressing a key question—how APA dysregulation contributes to TSC pathogenesis. Our results will also provide new insights into the knowledge and clinical management of TSC and other neurological disorders with mTOR pathway dysregulation (“mTORopathies”)