Identification of intrinsic and extrinsic regulators of TDP43 splicing function

项目摘要 TDP43剪接功能的内在和外在调节剂的鉴定。 痴呆症是一种毁灭性的和不可治愈的神经退行性疾病， 患者、他们的家人和世界各地的卫生系统。痴呆症的疾病改善疗法是 我们迫切需要，但由于我们缺乏对弱势群体的了解， 驱动其发病机制的分子网络。RNA结合蛋白TDP43的功能障碍已经出现， 在许多痴呆症中，包括阿尔茨海默病（AD）、边缘占主导地位的年龄相关痴呆症（AD）和阿尔茨海默病（AD）， TDP43脑病（LATE）和额颞叶痴呆（FTD）。TDP43作为蛋白质组的监护人 通过调节隐藏外显子（CE）的剪接，包含提前终止密码子的外显子， 移码突变，存在于数百种前mRNA中。然而，控制TDP43的监管网络 功能知之甚少，TDP43功能障碍导致神经变性的机制 仍然未知。为了帮助解决这一关键的知识差距，我们最近开发了一个定量的， 使用基于荧光的报告基因来测量单个活细胞中的TDP43依赖性剪接。结合这位记者 凭借我们在大规模全基因组筛选技术方面的专业知识，我们提出了两种筛选方法， 以全面和公正的方式绘制调节TDP43功能的分子网络。一是 将使用最近开发的深度突变扫描方法来确定TDP43所需的序列特征 用于其拼接功能。突变库将包含超过50，000个单点和双点突变， 在TDP43的固有无序结构域（IDR）上，由于IDR与两种剪接功能都有关系， 和聚合。为了鉴定调节TDP43依赖性剪接的反式作用因子和辅助因子，我们将 联合收割机将报告基因与基因组规模的功能丧失CRISPR筛选相结合。两个屏幕上的热门歌曲将 严格验证其对来源于以下的神经元中的TDP43依赖性剪接和聚集的作用： 小鼠胚胎干细胞这个项目的成功完成将揭示内在的IDR序列 直接或间接影响TDP43剪接功能的元件和外源性细胞组分， 从而为开发新的治疗策略所需的机制研究提供基础。

Project Summary Identification of intrinsic and extrinsic regulators of TDP43 splicing function. Dementias are devastating and incurable neurodegenerative diseases that represent an escalating burden on patients, their families and health systems throughout the world. Disease-modifying therapies for dementias are urgently needed, but their development has been hampered by our lack of knowledge about the vulnerable molecular networks that drive their pathogenesis. Dysfunction of the RNA-binding protein TDP43 has emerged pathological signature in many dementias, including Alzheimer’s disease (AD), limbic-predominant age-related TDP43 encephalopathy (LATE) and frontotemporal dementia (FTD). TDP43 functions as a guardian of proteome integrity by regulating the splicing of cryptic exons (CEs), exons that contain premature stop codons and frameshift mutations, present in hundreds of pre-mRNAs. However, the regulatory network that controls TDP43 function is poorly understood and the mechanism by which TDP43 dysfunction leads to neurodegeneration remains unknown. To help address this critical gap in knowledge, we have recently developed a quantitative, fluorescence-based reporter to measure TDP43-dependent splicing in single live cells. Combining this reporter with our expertise in large-scale, genome-wide screening technologies, we propose two screens designed to chart the molecular network that regulates TDP43 function in a comprehensive and unbiased manner. First, we will use recently developed deep mutational scanning methods to identify sequence features of TDP43 required for its splicing function. Mutant libraries will encompass over 50,000 single and double point mutations focused on the intrinsically disordered domain (IDR) of TDP43, since the IDR has been implicated in both splicing function and aggregation. To identify trans-acting factors and co-factors that regulate TDP43-dependent splicing, we will combine the reporter with genome-scale loss-of-function CRISPR screens. The top hits from both screens will be rigorously validated for their effects on TDP43-dependent splicing and aggregation in neurons derived from mouse embryonic stem cells. The successful completion of this project will uncover the intrinsic IDR sequence elements and extrinsic cellular components that directly or indirectly influence the splicing function of TDP43, thereby providing a foundation for the mechanistic studies required to develop new therapeutic strategies.