The Functional Role of RBM45 in Gene Expression and Neurodegeneration

RBM45 在基因表达和神经退行性变中的功能作用

• 批准号: 8398619
• 负责人: Mahlon Collins
• 金额: $ 4.22万
• 依托单位: ST. JOSEPH'S HOSPITAL AND MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-23 至 2015-05-22
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8398619
• 关键词: Address; Adult; Affect; Affinity; Alternative Splicing; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Apoptotic; Appearance; Arsenites; Binding; Biological Markers; Brain; Brain region; Cell Death; Cell Line; Cell Nucleus; Cell Survival; Cell physiology; Cells; Cerebrospinal Fluid; Cessation of life; Complex; Confocal Microscopy; Corticospinal Tracts; Cytoplasm; Cytoplasmic Granules; Data; Development; Diagnostic; Disease; Exons; FDA approved; Frequencies; Frontotemporal Lobar Degenerations; Gene Expression; Gene Expression Regulation; Gene Targeting; Goals; Hela Cells; Human; Immunohistochemistry; Immunoprecipitation; In Vitro; Investigation; Knowledge; Laboratories; Link; Liquid Chromatography; Location; Mean Survival Times; Measures; Mediating; Messenger RNA; Methods; Modification; Molecular; Morphology; Motor Neuron Disease; Motor Neurons; Nerve Degeneration; Neuraxis; Neurodegenerative Disorders; Normal Cell; Nuclear Localization Signal; Nuclear Translocation; Paralysed; Pathology; Pathway interactions; Patients; Phase; Phosphorylation; Poly C; Post-Translational Protein Processing; Process; Protein Isoforms; Proteins; Proteomics; RNA; RNA Binding; RNA Processing; RNA Splicing; RNA-Binding Proteins; Rattus; Recruitment Activity; Regulation; Research; Role; Series; Spinal Cord; Stress; Symptoms; Time; Tissues; Transcript; Translational Regulation; Ubiquitination; Variant; Western Blotting; base; biological adaptation to stress; cell type; crosslink; disorder control; insight; light microscopy; mRNA Expression; novel; overexpression; prognostic; protein TDP-43; research study; response; small hairpin RNA; tandem mass spectrometry

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a progressive, inevitably fatal neurodegenerative disorder. Pathologically, ALS is characterized by the death of motor neurons of the corticospinal tract, resulting in a numerous debilitating symptoms culminating with paralysis and death. Research into the molecular mechanisms of ALS neurodegeneration has implicated impaired RNA processing and intracytoplasmic aggregation of RNA binding proteins in cell death. We recently performed a cerebrospinal fluid (CSF)-based proteomics study of ALS patients and identified the novel RNA binding protein RBM45 as a putative disease biomarker. Further investigation of RBM45 via immunohistochemistry of ALS patient spinal cord tissue revealed RBM45-positive intracytoplasmic inclusions in motor neurons similar in appearance to those containing the other ALS-associated RNA binding proteins TDP-43 and FUS. We now seek to extend these preliminary observations by studying the normal function of RBM45 as well as its contribution(s) to neurodegenerative disease pathology. To do so, we will first assess how overexpression and knockdown of RBM45 influences the morphology, formation of inclusions, and viability of two cell lines. We will also overexpress a naturally-occurring isoform of the protein lacking a putative nuclear localization signal to determine the propensity of this variant to aggregate in vitro. The next goal of this proposal is t understand what role (if any) RBM45 has in the cellular response to stress. Cells will be stressed via treatment with arsenite and the morphology, number of RBM45-containing inclusions, and cell viability will again be measured. We will also examine the cells for changes in RBM45 post-translational modifications, colocalization with other RNA binding proteins, and incorporation into stress granules to address the function of RBM45 during stress. In the second phase of this proposal, RBM45 gene targets will be defined via UV cross-linking and immunoprecipitation in conjunction with RNA-Seq. We will next assess the contribution of RBM45 to the regulation of gene expression and alternative splicing. For these experiments, RBM45 will be depleted from cells via shRNA and the changes in gene expression and exon splicing will be determined using exon junction- sensitive microarrays. In the last phase of this proposal, we will attempt to link findings obtained in aims 1 and 2 to human neurodegenerative disease using human post-mortem tissue for immunohistochemistry and confocal microscopy. Tissues will be evaluated for brain region-specific and cell type-specific expression of RBM45, inclusions, and colocalization with other ALS- and stress response-associated proteins. Collectively, these experiments will provide new information about the role of RBM45 in normal cell function and disease. PUBLIC HEALTH RELEVANCE: ALS is the most common form of adult-onset motor neuron disease. The disorder is inevitably fatal, has a mean survival time of approximately 3 years, and only one FDA-approved treatment. Thus, there is a pressing need for new insights into the mechanisms of this illness.

描述（由申请人提供）：肌萎缩侧索硬化症（ALS）是一种进行性的、不可避免的致命性神经退行性疾病。从病理学上来说，ALS 的特点是皮质脊髓束运动神经元死亡，导致多种衰弱症状，最终导致瘫痪和死亡。对 ALS 神经变性分子机制的研究表明，细胞死亡过程中 RNA 加工受损以及 RNA 结合蛋白的胞质内聚集。我们最近对 ALS 患者进行了一项基于脑脊液 (CSF) 的蛋白质组学研究，并确定了新型 RNA 结合蛋白 RBM45 作为假定的疾病生物标志物。通过 ALS 患者脊髓组织的免疫组织化学对 RBM45 进行进一步研究，发现运动神经元中的 RBM45 阳性胞质内包涵体在外观上与含有其他 ALS 相关 RNA 结合蛋白 TDP-43 和 FUS 的包涵体相似。我们现在寻求通过研究 RBM45 的正常功能及其对神经退行性疾病病理学的贡献来扩展这些初步观察结果。为此，我们将首先评估 RBM45 的过表达和敲低如何影响两种细胞系的形态、内含物的形成和活力。我们还将过度表达缺乏假定的核定位信号的天然存在的蛋白质亚型，以确定该变体在体外聚集的倾向。该提案的下一个目标是了解 RBM45 在细胞对压力的反应中起什么作用（如果有的话）。将通过亚砷酸盐处理对细胞施加压力，并再次测量含有 RBM45 的内含物的形态、数量和细胞活力。我们还将检查细胞中 RBM45 翻译后修饰的变化、与其他 RNA 结合蛋白的共定位以及掺入应激颗粒中以解决 RBM45 在应激期间的功能。在该提案的第二阶段，将通过 UV 交联和免疫沉淀结合 RNA-Seq 来定义 RBM45 基因靶标。接下来我们将评估 RBM45 对基因表达和选择性剪接调节的贡献。对于这些实验，将通过shRNA从细胞中耗尽RBM45，并且将使用外显子连接敏感微阵列来确定基因表达和外显子剪接的变化。在该提案的最后阶段，我们将尝试使用人类死后组织进行免疫组织化学和共聚焦显微镜检查，将目标 1 和 2 中获得的发现与人类神经退行性疾病联系起来。将评估组织中 RBM45 的大脑区域特异性和细胞类型特异性表达、内含物以及与其他 ALS 和应激反应相关蛋白的共定位。总的来说，这些实验将提供有关 RBM45 在正常细胞功能和疾病中作用的新信息。 公共卫生相关性：ALS 是成人发病的运动神经元疾病的最常见形式。这种疾病不可避免地是致命的，平均生存时间约为 3 年，而且只有一种 FDA 批准的治疗方法。因此，迫切需要对这种疾病的机制有新的见解。