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。

基本信息

批准号：
10813541
负责人：
Laxman Dass Gangwani
金额：
$ 42.14万
依托单位：
UNIVERSITY OF MISSOURI-COLUMBIA
依托单位国家：
美国
项目类别：
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/10813541
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

项目摘要

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.

项目摘要这项研究的目的是检查锌指蛋白ZPR1在R环代谢和神经变性。在转录过程中形成R环，由RNA-DNA杂交链和A组成互补的DNA链。 R环的积累导致DNA损伤导致神经变性与遗传神经退行性疾病有关，包括脊柱肌肉萎缩（SMA）和肌萎缩症侧硬化症（ALS）。 ZPR1在真核生物中是进化保守的，对于细胞活力是必不可少的。然而，关于可能有助于细胞活力和人类疾病的ZPR1生物学功能知之甚少病因。 ZPR1与翻译伸长因子1a（EF1A），RNA聚合酶II和鼻纳抗素直接相互作用（SETX），在真核生物中也保守。 SETX是分辨R-所需的RNA-DNA解旋酶循环。 SETX中的突变与一组不可治疗的神经退行性疾病有关，包括动脉桥眼动作2型，常染色体显性sma和ALS4，其特征是R环中的缺陷代谢。 R环分辨率的分子机制在很大程度上未知。我们的初步数据显示 ZPR1缺乏会导致R环的积累和神经变性。 ZPR1过表达减少R- 循环并营救神经元和患者细胞中的DNA损伤，并防止SMA小鼠的神经退行性。 ZPR1与RNA-DNA杂种并与体内的R-Loops结合。 zpr1与setx和zpr1互动用R环形成SETX复合物所必需的，这表明ZPR1可能有助于招募SETX到R- 循环。我们创建了新颖的ZPR1突变小鼠，ZPR1基因座的双重和四倍点突变为有选择地破坏ZPR1-EF1A复合物。 ZPR1突变小鼠显示R环的积累并发展神经退行性疾病样的表型类似于SETX突变患者的报道。一起，这些发现提出了一个假设，即具有EF1A和SETX的ZPR1复合物可能起独特而关键的作用在R-loop分辨率中，为研究ZPR1-EF1A和ZPR1-SETX的功能提供了基础 R环代谢中的复合物。具体目的是检查：（目标1）ZPR1-的分子基础使用ZPR1条件小鼠共转录R环和神经退行性的依赖性积累；（目的 2）使用新型鼠标模型破坏ZPR1-EF1A，ZPR1-EF1A复合物的功能在R环分辨率中的功能我们已经生成的运动神经元中的体内复合物，以及GTP/GDP依赖的机理 ZPR1-EF1A复合物对RNA和DNA链的分辨率；（AIM 3）ZPR1-SETX复合物的功能在R环代谢，基因组完整性和ALS4发病机理中。 ZPR1-固定复合物在ALS4中被破坏 SETX突变的患者。 ZPR1-setx复合物破坏对R环代谢，DNA的影响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。这项研究将提供对R环代谢缺陷引起的发病机理的分子基础的全面洞察力，将为一组无法治愈的疾病制定有针对性的治疗策略的突破。