The functions of the zinc finger protein ZPR1 in R-loop metabolism and neurodegeneration.

锌指蛋白 ZPR1 在 R 环代谢和神经退行性变中的功能。

• 批准号: 10548749
• 负责人: Laxman Dass Gangwani
• 金额: --
• 依托单位: TEXAS TECH UNIVERSITY HEALTH SCIENCES CENTER AT EL PASO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2023-01-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10548749
• 关键词: Adenoviruses; Alleles; Amyotrophic Lateral Sclerosis; Apraxias; Ataxia; Binding; Binding Proteins; Biochemical; Biological; Biological Process; Cell Survival; Cells; Complementary DNA; Complex; Coupled; DNA; DNA Damage; DNA Polymerase II; DNA Repair; DNA biosynthesis; DNA replication fork; Data; Defect; Disease; Elongation Factor; Etiology; Eukaryota; Foundations; Gel; Genes; Genetic; Genetic Transcription; Genomic Instability; Goals; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hybrids; Hydrolysis; In Vitro; LoxP-flanked allele; Mediating; Metabolism; Methods; Modeling; Molecular; Molecular Target; Motor Neurons; Mus; Mutant Strains Mice; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neuromuscular Diseases; Neurons; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Phenotype; Physiological; Play; Point Mutation; Proteins; RNA; RNA Polymerase II; Reporting; Resolution; Role; SMN protein (spinal muscular atrophy); SMN1 gene; Spinal Muscular Atrophy; System; Testing; Therapeutic; Tissues; Translations; Yeast Model System; Yeasts; Zinc Fingers; autosome; chromatin immunoprecipitation; experimental study; genome integrity; helicase; human disease; in vivo; insight; mouse model; novel; oculomotor; overexpression; postmitotic; prevent; recruit; response; targeted treatment; transcriptome; transcriptome sequencing

PROJECT SUMMARY The goal of this study is to examine the functions of the zinc finger protein ZPR1 in R-loops metabolism and neurodegeneration. R-loops are formed during transcription and consist of RNA-DNA hybridized strands and a complementary DNA strand. R-loop accumulation results in DNA damage leading to neurodegeneration associated with genetic neurodegenerative diseases, including spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). ZPR1 is evolutionary conserved in eukaryotes and is essential for cell viability. However, very little is known about the ZPR1 biological functions that may contribute to cell viability and human disease etiology. ZPR1 interacts directly with translation elongation factor 1A (EF1A), RNA Polymerase II and Senataxin (SETX), which are also conserved in eukaryotes. SETX is an RNA-DNA helicase required for resolution of R- loops. Mutations in SETX are associated with a group of untreatable neurodegenerative disorders, including ataxia oculomotor apraxia type 2, autosomal dominant SMA and ALS4, characterized by defects in R-loop metabolism. The molecular mechanisms of R-loop resolution are largely unknown. Our preliminary data show that ZPR1 deficiency causes R-loop accumulation and neurodegeneration. ZPR1 overexpression reduces R- loops and rescues DNA damage in neurons and patient cells, and prevents neurodegeneration in SMA mice. ZPR1 binds to RNA-DNA hybrids and associates with R-loops in vivo. ZPR1 interacts with SETX and ZPR1 is required for the formation of SETX complexes with R-loops suggesting that ZPR1 may help recruit SETX to R- loops. We have created novel Zpr1 mutant mice, with double and quadruple point mutations in the Zpr1 locus to selectively disrupt ZPR1-EF1A complexes. ZPR1 mutant mice show accumulation of R-loops and develop neurodegenerative disease-like phenotypes similar to reported for patients with SETX mutations. Together, these findings raise a hypothesis that ZPR1 complexes with EF1A and SETX may play distinct and critical roles in R-loop resolution and provide a foundation for investigating the function of ZPR1-EF1A and ZPR1-SETX complexes in R-loop metabolism. The specific aims are to examine: (Aim 1) the molecular basis of ZPR1- dependent accumulation of co-transcriptional R-loops and neurodegeneration using Zpr1 conditional mice; (Aim 2) the function of ZPR1-EF1A complexes in R-loop resolution using novel mouse models to disrupt ZPR1-EF1A complexes in vivo in motor neurons that we have generated, and the mechanism of GTP/GDP-dependent resolution of RNA and DNA strands by ZPR1-EF1A complexes; (Aim 3) the function of ZPR1-SETX complexes in R-loop metabolism, genome integrity and ALS4 pathogenesis. ZPR1-SETX complexes are disrupted in ALS4 patients with SETX mutation. The effect of disruption of ZPR1-SETX complexes on R-loop metabolism, DNA replication fork and genome integrity using cell-based models, including patient cells. This study will provide comprehensive insight into the molecular basis of pathogenesis caused by defects in R-loop metabolism that would be a breakthrough towards developing targeted therapeutic strategies for a group of incurable diseases.

项目总结 本研究的目的是研究锌指蛋白ZPR1在R-环代谢和蛋白质合成中的作用。 神经退行性变。R-环是在转录过程中形成的，由RNA-DNA杂交链和 互补的DNA链。R环堆积导致DNA损伤导致神经变性 与遗传性神经退行性疾病有关，包括脊髓性肌萎缩(SMA)和肌营养不良 侧索硬化症(ALS)ZPR1在真核生物中是进化保守的，对细胞活力是必不可少的。然而， 人们对ZPR1的生物学功能知之甚少，这些功能可能有助于细胞存活和人类疾病 病因学。ZPR1与翻译延长因子1A(EF1A)、RNA聚合酶II和感觉神经毒素直接相互作用 (SETX)，它们在真核生物中也是保守的。SETX是一种RNA-DNA解旋酶，需要拆分R- 循环。SETX基因突变与一组无法治疗的神经退行性疾病有关，包括 共济失调动眼运动性失用症2型，常染色体显性遗传性SMA和ALS4，以R环缺陷为特征 新陈代谢。R-环拆分的分子机制在很大程度上是未知的。我们的初步数据显示 ZPR1缺乏会导致R环堆积和神经退行性变。ZPR1过表达降低R- 循环并挽救神经元和患者细胞中的DNA损伤，防止SMA小鼠的神经退化。 在体内，ZPR1与RNA-DNA杂交物结合，并与R-环结合。ZPR1与SETX相互作用，ZPR1是 与R-环形成SETX复合体所必需的，提示ZPR1可能有助于将SETX招募到R- 循环。我们已经创造了新的Zpr1突变小鼠，在Zpr1基因座上具有双点和四点突变，以 选择性地破坏ZPR1-EF1A复合体。ZPR1突变小鼠表现出R-环的积累和发育 神经退行性疾病样表型类似于SETX突变患者的报道。一起， 这些发现提出了一种假设，即ZPR1与EF1A和SETX的复合体可能发挥着不同的关键作用 为进一步研究ZPR1-EF1A和ZPR1-SETX的功能奠定了基础 R-环代谢中的复合体。具体目的是研究：(目标1)ZPR1-的分子基础。 使用Zpr1条件性小鼠依赖共转录R-环的积累和神经变性；(目的 2)利用新型小鼠模型研究ZPR1-EF1A复合体在R环分辨中的作用 我们产生的运动神经元体内复合体以及GTP/GDP依赖的机制 ZPR1-EF1A络合物拆分RNA和DNA链；(目标3)ZPR1-SETX络合物的功能 在R环代谢、基因组完整性和ALS4发病机制中的作用。ZPR1-SETX复合体在ALS4中被破坏 携带SETX突变的患者。ZPR1-SETX复合体破坏对R环代谢、DNA的影响 复制分叉和基因组完整性使用基于细胞的模型，包括患者细胞。这项研究将提供 全面了解R环代谢缺陷引起的发病的分子基础 这将是为一组不治之症制定有针对性的治疗战略的一个突破。