Functional Post-Transcriptional Interplay of the Nuclear RNA Gomafu During Human Neural Cell Development

人类神经细胞发育过程中核 RNA Gomafu 的功能性转录后相互作用

• 批准号: 10314746
• 负责人: Paul Michael ZAKUTANSKY
• 金额: $ 4.6万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10314746
• 关键词: Affect; Alternative Splicing; Autopsy; Binding; Biogenesis; Biological Assay; Brain; Brain Diseases; CRISPR/Cas technology; Cell Line; Cell Nucleus; Cell model; Clip; Cognition Disorders; Cognitive; Complex; Data; Development; Disease; Etiology; Exclusion; Exons; Gene Expression Regulation; Genetic Transcription; Goals; Human; Immunoblot Analysis; Immunofluorescence Immunologic; In Vitro; Knock-out; Knowledge; Liquid Chromatography; Mental disorders; Messenger RNA; Molecular; Mutation; Myocardial Infarction; Names; Neurodegenerative Disorders; Neuroglia; Neuronal Differentiation; Neurons; Nuclear; Nuclear RNA; Nucleotides; Pathogenesis; Pathway interactions; Patients; Play; Population; Process; Protein Isoforms; Protein Splicing; Proteins; RNA Binding; RNA Splicing; RNA-Binding Proteins; Recombinants; Regulation; Reporting; Response Elements; Reverse Transcriptase Polymerase Chain Reaction; Risk Factors; Role; Schizophrenia; Stem Cell Development; Testing; Transcript; Untranslated RNA; Up-Regulation; Western Blotting; first episode schizophrenia; induced pluripotent stem cell; insight; interest; mRNA Precursor; nerve stem cell; neuron development; neuropsychiatric disorder; neuropsychiatry; novel; novel strategies; schizophrenia risk; stem cell function; tandem mass spectrometry

PROJECT SUMMARY Many cognitive disorders arise from abnormalities at the early stage of brain development. Alternative splicing, the inclusion or exclusion of specific exons in mRNAs, is a key component in controlling normal neuronal development but affected in neuropsychiatric diseases represented by schizophrenia (SCZ). Recent discoveries indicate that long non-coding RNAs (lncRNAs), a class of non-coding RNAs longer than 200 nucleotides which do not encode for proteins, play sophisticated roles in gene regulation including alternative splicing. IncRNAs are poorly conserved in general and highly expressed in the human brain. Abnormalities in lncRNA expression are implicated in neurodegenerative and neuropsychiatric diseases. A particular human lncRNA of interest is GOMAFU, which is abundantly expressed in human iPSC-derived neural progenitor cells (hNPCs) and brain neurons but negligible in glia. GOMAFU is a nuclear lncRNA affected in SCZ and known to regulate alternative splicing of a number of risk factor transcripts involved in SCZ. However, molecular mechanisms regulating GOMAFU are undefined. Moreover, although GOMAFU was postulated to regulate alternative splicing through sequestering neuronal RNA-binding protein (RBP) splicing factors, RBPs interacting with GOMAFU still remain elusive. Emerging evidence, including our preliminary data, indicates that GOMAFU may form a functional pathway with an RBP called Quaking I (QKI), another SCZ risk factor known to regulate alternative splicing in neural progenitor cells (NPCs) and neuron-glia lineage development. Recombinant QKI was shown to interact with GOMAFU in vitro. Furthermore, during development of human iPSC-derived cortical neurons, the decline of QKI conversely associates with increased GOMAFU expression. Importantly, elimination of QKI leads to GOMAFU up-regulation in a human NPC cell line. Thus, I hypothesize that QKI-5 suppresses GOMAFU expression in human NPCs whereas GOMAFU controls splicing in human neuron development through sequestration of nuclear splicing factors, including QKI-5. The goal of this project is to delineate the function of the QKI-GOMAFU SCZ risk factor pathway in alternative splicing during human neuronal development. In Aim 1, I will determine whether QKI-5 binds and suppresses GOMAFU biogenesis in a hNPC cell line and human iPSC-derived NPCs. In Aim 2, I will determine whether GOMAFU regulates QKI nuclear distribution and splicing function in hNPCs. Moreover, I will utilize the recently developed comprehensive identification of RNA binding proteins (ChIRP) assay to identify GOMAFU-bound RBP splicing factors and elucidate how GOMAFU modulate their function in alternative splicing during hNPC development.

项目摘要 许多认知障碍是由大脑发育早期的异常引起的。选择性剪接， 在mRNA中包含或排除特定外显子是控制正常神经元 但在以精神分裂症（SCZ）为代表的神经精神疾病中受到影响。最近的发现 表明最长非编码RNA（lncRNA），一类长度超过200个核苷酸的非编码RNA， 不编码蛋白质，在基因调控中发挥复杂的作用，包括选择性剪接。的incrna 一般保守性差，在人脑中高度表达。lncRNA表达异常 与神经变性和神经精神疾病有关。感兴趣的特定人lncRNA是 GOMAFU，其在人iPSC衍生的神经祖细胞（hNPC）和脑中大量表达， 神经元，但在胶质细胞中可忽略不计。GOMAFU是一种在SCZ中受影响的核lncRNA，已知可调节替代性 SCZ中涉及的许多危险因子转录本的剪接。然而，调节的分子机制 GOMAFU未定义。此外，虽然GOMAFU被假定通过调节选择性剪接， 隔离神经元RNA结合蛋白（RBP）剪接因子，与GOMAFU相互作用的RBP仍然存在 难以捉摸。新出现的证据，包括我们的初步数据，表明GOMAFU可能形成一个功能性的， RBP途径称为Quaking I（QKI），这是另一种已知调节选择性剪接的SCZ风险因素， 神经祖细胞（NPC）和神经胶质细胞谱系发育。重组QKI显示出相互作用 GOMAFU在体外此外，在人类iPSC衍生的皮层神经元的发育过程中， 相反，QKI的表达与GOMAFU表达的增加相关。重要的是，消除QKI导致 人NPC细胞系中的GOMAFU上调。因此，我假设QKI-5抑制GOMAFU， 而GOMAFU控制人类神经元发育中的剪接， 核剪接因子的螯合，包括QKI-5。这个项目的目标是描绘功能 QKI-GOMAFU SCZ风险因子通路在人类神经元发育过程中的选择性剪接。在Aim中 1中，我将确定QKI-5是否结合并抑制hNPC细胞系和人类中的GOMAFU生物合成。 iPSC衍生的NPC。在目标2中，我将确定GOMAFU是否调节QKI核分布和剪接 在hNPC中发挥作用。此外，我将利用最近开发的RNA结合的综合鉴定 蛋白质（ChIRP）测定，以鉴定GOMAFU结合的RBP剪接因子，并阐明GOMAFU如何调节 它们在hNPC发育过程中的可变剪接中的功能。