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: 10323049
• 负责人: Laxman Dass Gangwani
• 金额: $ 42.43万
• 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 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10323049
• 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; Mitotic; 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; base; chromatin immunoprecipitation; experimental study; genome integrity; helicase; human disease; in vivo; insight; mouse model; novel; oculomotor; overexpression; 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.

项目摘要 本研究的目的是检测锌指蛋白ZPR 1在R环代谢中的功能， 神经变性R环是在转录过程中形成的，由RNA-DNA杂交链和RNA链组成。 互补DNA链。R环积累导致DNA损伤，导致神经退行性变 与遗传性神经退行性疾病相关，包括脊髓性肌萎缩症（SMA）和肌萎缩性 侧索硬化症（ALS）。ZPR 1在真核生物中是进化保守的，并且对于细胞活力是必需的。然而，在这方面， 关于ZPR 1的生物学功能，可能有助于细胞活力和人类疾病的了解很少 病因学ZPR 1与翻译延伸因子1A（EF 1A）、RNA聚合酶II和Senataxin直接相互作用 （SETX），其在真核生物中也是保守的。SETX是一种RNA-DNA解旋酶，用于拆分R- 循环SETX的突变与一组无法治疗的神经退行性疾病有关，包括 共济失调眼运动不能2型，常染色体显性SMA和ALS 4，以R环缺陷为特征 新陈代谢. R环分解的分子机制在很大程度上是未知的。我们的初步数据显示 ZPR 1缺陷导致R环积聚和神经退行性变。ZPR 1过表达减少了R- 循环和挽救神经元和患者细胞中的DNA损伤，并防止SMA小鼠的神经变性。 ZPR 1结合RNA-DNA杂合体，并在体内与R环结合。ZPR 1与SETX相互作用，ZPR 1是 这表明ZPR 1可能有助于将SETX募集到R-环上。 循环我们创造了新型Zpr 1突变小鼠，其Zpr 1基因座具有双重和四重点突变， 选择性破坏ZPR 1-EF 1A复合物。ZPR 1突变小鼠显示R环的积累并发展为 神经退行性疾病样表型与SETX突变患者报告的相似。在一起， 这些发现提出了一个假设，即ZPR 1与EF 1A和SETX的复合物可能发挥不同的关键作用 为进一步研究ZPR 1-EF 1A和ZPR 1-SETX的功能奠定了基础 R环代谢中的复合物。具体的目的是检查：（目的1）ZPR 1的分子基础- 使用Zpr 1条件小鼠的共转录R环和神经变性的依赖性积累;（目的 2)使用新型小鼠模型破坏ZPR 1-EF 1A，ZPR 1-EF 1A复合物在R环解析中的功能 复合物在体内的运动神经元，我们已经产生，和GTP/GDP依赖的机制， ZPR 1-EF 1A复合物对RNA和DNA链的拆分;（目的3）ZPR 1-SETX复合物的功能 在R环代谢、基因组完整性和ALS 4发病机制中的作用。ZPR 1-SETX复合物在ALS 4中被破坏 SETX突变患者。ZPR 1-SETX复合物的破坏对R环代谢、DNA 复制叉和基因组完整性使用基于细胞的模型，包括患者细胞。本研究将提供 全面深入了解R环代谢缺陷引起的发病机制的分子基础， 将是一个突破，为一组不治之症制定有针对性的治疗策略。