The role of poison exons in neurodevelopment

毒外显子在神经发育中的作用

• 批准号: 10373264
• 负责人: Gemma Louise Carvill
• 金额: $ 44万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2024-03-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10373264
• 关键词: Alternative Splicing; Biological Assay; Brain; Cell Cycle; Cell Line; Cell Proliferation; Cells; Complement; Cortical Malformation; Coupled; Cycloheximide; DNA; Data; Deoxyuridine; Development; Disease; Ectopic Expression; Elements; Epilepsy; Excision; Exons; Funding; Future; Gene Expression; Genes; Genome; Guide RNA; Human; Human Development; Individual; Integral Membrane Protein; Ion Transport; Ions; Lead; Libraries; Mitosis; Mitotic; Modeling; Mus; Nature; Neurodevelopmental Disorder; Neuronal Differentiation; Neurons; Nonsense-Mediated Decay; Outcome Study; Poison; Production; Protein Isoforms; Proteins; RNA Splicing; Research Proposals; Role; Synaptic Vesicles; Technology; Terminator Codon; Testing; Transcript; base; cell motility; cell type; course development; design; excitatory neuron; genetic analysis; genetic variant; genome sequencing; high reward; high risk; high throughput screening; induced pluripotent stem cell; inhibitor/antagonist; nerve stem cell; nervous system disorder; neurodevelopment; neurogenetics; neuron development; neuron loss; neuropsychiatric disorder; novel; premature; progenitor; programs; transcriptome sequencing; vesicular release

Project summary Exons in the genome that lead to the introduction of premature truncation (stop) codon (PTC) and mark these transcripts as targets for nonsense-mediated decay (NMD) are called poison exons (PEs). These PEs have recently been shown to be used throughout mouse and human neurodevelopment where they are alternatively spliced over the course of development. Moreover, genetic variants that perturb the splicing of PEs have been associated with neurodevelopmental disorders, including epilepsy and malformations of cortical development. However, the full complement of PEs that are alternatively spliced throughout human development are not known. Therefore, in Aim 1 of this study we will use long-read sequencing in induced pluripotent stem cell (iPSC)-derived neuronal progenitor cells (NPCs) and neurons, treated with cycloheximide to inhibit NMD, to identify PEs. Moreover, while recent efforts have elucidated the use of PEs during neurodevelopment, we have very limited understanding of their function. In Aim 2 we will adapt the recently described paired guide RNAs for alternative exon removal (pgFARM) approach to delete candidate PEs in iPSC-derived neuronal progenitor cells (NPCs) and determine the effects on proliferation of these cells. Our preliminary analysis suggests that many PEs that are included in transcripts in NPCs (NPC-PEs), encode for genes required for mature neuronal function, including ion transport and synaptic vesicle assembly and release. We hypothesize that deletion of NPC-PEs will lead to the ectopic expression of these genes, exit of the cell-cycle and premature neuronal differentiation of NPCs. This exploratory, high-risk, high-reward study is not intended to identify all PEs that are alternatively spliced throughout development, rather we aim to determine the efficacy of using long-read sequencing where NMD is inhibited to identify PEs. Moreover, we aim to develop a high-throughput assay for determining the role of these PEs in early stages of neurodevelopment. Collectively, the outcomes of this study will provide the platforms and experimental paradigms for (1) identifying PEs used in human neurodevelopment (2) provide a framework for the interpretation of intronic genetic variants identified by genome sequencing in individuals with neurodevelopmental and neuropsychiatric disorders (3) determining the function of these PEs in development. We intend that this data will be used to pursue future individual or program level funding applications.

项目摘要 基因组中导致引入过早截短（终止）密码子（PTC）并标记这些 作为无义介导的衰变（NMD）的靶的转录物被称为毒物外显子（PE）。这些PE具有 最近被证明在小鼠和人类神经发育中使用， 在发展过程中拼接而成。此外，已经发现了干扰PE剪接的遗传变异 与神经发育障碍相关，包括癫痫和皮质发育畸形。 然而，在整个人类发育过程中选择性剪接的PE的完整补充并不 知道的因此，在本研究的目的1中，我们将在诱导多能干细胞中使用长读段测序， 用放线菌酮处理以抑制NMD的iPSC衍生的神经元祖细胞（NPC）和神经元， 识别PE。此外，虽然最近的努力已经阐明了PE在神经发育过程中的作用，但我们 对其功能的了解非常有限。在目标2中，我们将调整最近描述的成对引导RNA 选择性外显子去除（pgFARM）方法删除iPSC衍生的神经元祖细胞中的候选PE 细胞（NPC），并确定对这些细胞增殖的影响。我们的初步分析表明， 许多包含在NPC转录物中的PE（NPC-PE）编码成熟神经元生长所需的基因， 功能，包括离子转运和突触囊泡组装和释放。我们假设， NPC-PE可导致这些基因的异位表达，细胞周期的退出和神经元的早熟 NPC的分化。这项探索性、高风险、高回报的研究并非旨在识别所有 在整个开发过程中选择性剪接，而我们的目标是确定使用长读 测序，其中NMD被抑制以鉴定PE。此外，我们的目标是开发一种高通量的检测方法， 确定这些PE在神经发育早期阶段的作用。总的来说，这项研究的结果 将提供平台和实验范例，用于（1）鉴定用于人类的PE 神经发育（2）提供了一个框架，用于解释内含子遗传变异， 神经发育和神经精神障碍个体的基因组测序（3）确定 在发展过程中发挥这些作用。我们打算将这些数据用于未来的个人或 项目级资金申请。