Investigating RNA dysregulation in Neurological Disease through study of Pontocerebellar Hypoplasia Type 1b

项目摘要/摘要 桥小脑发育不全1b型(PCH1B)是一种常染色体隐性遗传性神经系统疾病，其特征是 小脑和脑桥发育不良/萎缩，通常在出生后一年内致命。小脑和 脑桥整合来自感觉系统、脊髓和大脑其他部分的信息来调节运动 运动、呼吸和学习运动行为。PCH1B携带者表现为肌肉萎缩/虚弱， 小头畸形和发育迟缓。大多数PCH1B携带者没有活过童年和目前 治疗完全是治标不治本。导致PCH1B的突变发生在EXOSC3基因，该基因编码 进化上保守且普遍表达的RNA加工复合体的结构帽亚单位， 核糖核酸外切体。RNAexosome是一种核糖核酸酶，由结构亚基和催化亚基组成 在RNA转录后调控中起关键作用。这个复合体是3‘到5’处理所必需的 核内和细胞质中大量RNA的降解。转录后加工 RNA是基因表达的关键调控步骤，神经系统疾病的数量突显了这一点 由RNA加工因子的缺陷引起。RNA外切体引起的组织特异性表型 基于目前的RNA外切体功能模型，复杂的理解具有挑战性 分析体内任何多细胞模型中的复合体。因此，我们的目标是研究体内功能 EXOSC3中不同致病氨基酸替换与一系列温和的 PCH1B的严重表型。我们已经产生了一系列与EXOSC3疾病相关的错义等位基因 利用CRISPR/Cas9编辑技术对果蝇同源基因Rrp40进行突变。我们之前在苍蝇上的工作 揭示了神经元对Rrp40的需求增强。此外，我们对大脑富集物的RNA-seq分析 Rrp40突变体的转录本显示功能重要神经元的稳态水平增加 转录本，表明果蝇RNA外切体亚单位Rrp40中致病氨基酸的变化 会导致神经元功能障碍。我们现在的目标是确定致病的氨基酸替换是如何 在Rrp40中，改变果蝇体内发育中的神经系统的分子和细胞景观。我们 将检验这样一种假设，即RNA外切体调节对正常 神经发育和功能，其中一个不同的子集由RNA外切体帽调节 Rrp40亚基(EXOSC3)通过三个互补的目的：1)评估氨基的功能后果 果蝇RNA外切体亚单位Rrp40中与导致PCH1B相应的酸取代；2) 询问Rrp40突变如何影响果蝇体内关键神经元转录本的表达和定位 大脑；以及3)利用基因筛查来确定特定RNA外切体的异常积累 靶标会破坏苍蝇的发育和/或动态平衡。这些目标的成功实现将提供一个 协同理解RNA外切体生物学和对PCH1B疾病病因学的宝贵见解。

PROJECT SUMMARY/ABSTRACT Pontocerebellar Hypoplasia Type 1b (PCH1b) is an autosomal recessive neurological disorder characterized by hypoplasia/atrophy of the cerebellum and pons that is often fatal within the first year of life. The cerebellum and pons integrate information from sensory systems, the spinal cord, and other parts of the brain to regulate motor movements, breathing, and learning motor behavior. Individuals with PCH1b show muscle atrophy/weakness, microcephaly, and developmental delay. Most individuals with PCH1b do not live past childhood and current treatment is purely palliative. Mutations that cause PCH1b occur in the EXOSC3 gene, which encodes a structural cap subunit of an evolutionarily conserved and ubiquitously expressed RNA processing complex, the RNA exosome. The RNA exosome is a ribonuclease composed of both structural and catalytic subunits that play a critical role in the post-transcriptional regulation of RNA. This complex is required for 3’ to 5’ processing and degradation of a vast number of RNAs in both the nucleus and cytoplasm. Post-transcriptional processing of RNA is a critical regulatory step in gene expression, as underscored by the number of neurological diseases caused by defects in RNA processing factors. The tissue-specific phenotypes caused by the RNA exosome complex are challenging to understand based on current models of RNA exosome function with only limited analysis of the complex in any multicellular model in vivo. Thus, we aim to investigate the in vivo functional consequences of distinct disease-causing amino acid substitutions in EXOSC3 that are linked to a range of mild to severe phenotypes in PCH1b. We have generated an allelic series of EXOSC3 disease-linked missense mutations in the Drosophila orthologue Rrp40 via CRISPR/Cas9 editing technology. Our previous work in flies revealed an enhanced requirement for Rrp40 in neurons. Furthermore, our RNA-seq analysis of brain-enriched transcriptomes of Rrp40 mutants revealed increases in steady-state levels of functionally important neuronal transcripts, suggesting that disease-causing amino acid changes in the Drosophila RNA exosome subunit Rrp40 contribute to neuronal dysfunction. Our goal now is to characterize how disease-causing amino acid substitutions in Rrp40 alter the molecular and cellular landscape of the developing nervous system in Drosophila in vivo. We will test the hypothesis that the RNA exosome regulates RNAs that are critical for proper neurodevelopment and function, a distinct subset of which are regulated by the RNA exosome cap subunit Rrp40 (EXOSC3) through three complementary aims: 1) Assess the functional consequences of amino acid substitutions in the Drosophila RNA exosome subunit Rrp40 corresponding to those that cause PCH1b; 2) Interrogate how Rrp40 mutations affect expression and localization of key neuronal transcripts within the fly brain; and 3) Exploit a genetic screen to determine whether aberrant accumulation of specific RNA exosome targets disrupt fly development and/or homeostasis. Successful completion on these aims will provide a synergistic understanding of RNA exosome biology and invaluable insights into PCH1b disease etiology.