Regulation and biological functions of mRNA Alternative Polyadenylation in the Brain

项目摘要 选择性多聚腺苷(APA)在大脑中产生高度多样化的转录组和蛋白质组，以 保持适当的神经功能。其他人和我们最近的作品揭示了一种复杂和动态的 大脑中的APA景观与许多细胞类型特异的APA调节和APA在 神经系统疾病。然而，神经疾病中APA的改变在很大程度上仍未被探索和研究。 严格设计的体内高通量APA脑部疾病研究尤其缺乏，因为 技术上的困难。2017年，我们通过开发CTAG-回形针技术打破了技术壁垒， 它同时分析了来自完整小鼠大脑的单个细胞类型中的20,000多个聚(A)位点 并且比其他需要细胞纯化的测序策略更好地保存体内转录组。 结节性硬化症(TSC)的特点是脑内mTORC1信号的激活，严重的 神经症状。有趣的是，mTORC1的激活最近被证明能引起全球近端 通过调节非脑细胞中的APA因子使APA漂移，这表明异常的APA是一种 先前未知的TSC的细胞表型。在本申请中，我们建议进行一项证明- 应用CTAG-回形针结合新工具研究TSC中APA的原理研究 这是我们最近开发的。我们将确定APA亚型表达的变化，以响应急性丢失的 通过体内CTAG-回形针分析3种脑细胞类型的TSC1和mTORC1信号的激活： 兴奋性神经元、抑制性神经元和星形胶质细胞。我们将利用我们已建立的生物信息学渠道 确定每种细胞类型中稳健且具有生物学意义的APA变化。我们将执行RNA-seq to 为观察到的APA变化提供独立验证。APA可以改变蜂窝信令网络 通过产生截短的蛋白质或失去RNA结合蛋白的3‘UTR调控，以及 MicroRNA。对于预测将带来这种影响的APA变化，我们将通过实验验证预测 普通细胞系和原代神经元培养中蛋白质的定量或定性变化。最后，我们将 同时还探讨了线粒体TORC1激活引起APA变化的分子机制。 原代神经元培养。在拟议的项目完成后，我们预计将首次披露如何 TSC中mTORC1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-APA失调如何促进TSC 发病机制。我们的研究结果也将为认识和临床管理提供新的见解。 TSC和其他伴有mTOR通路失调的神经疾病(“mTORopathies”)

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”)