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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

基本信息

批准号：
8580899
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
项目类别：
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/8580899
关键词：
Address Adult Affect Affinity Alternative Splicing Alzheimer&apos 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 public health relevance research study response small hairpin RNA tandem mass spectrometry transcriptome sequencing

项目摘要

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.

描述（由申请人提供）：肌萎缩侧索硬化症（ALS）是一种进行性、不可避免的致命性神经退行性疾病。在病理学上，ALS的特征在于皮质脊髓束的运动神经元的死亡，导致许多衰弱症状，最终导致瘫痪和死亡。对ALS神经变性的分子机制的研究表明，细胞死亡中RNA加工受损和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，并确定了新的RNA结合蛋白RBM 45作为推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RBM 45的进一步研究显示，运动神经元中的RBM 45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的那些类似。我们现在试图通过研究RBM 45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。为此，我们将首先评估RBM 45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。我们还将过表达缺乏推定的核定位信号的蛋白质的天然存在的同种型，以确定该变体在体外聚集的倾向。这个提议的下一个目标是了解RBM 45在细胞对压力的反应中有什么作用（如果有的话）。将通过用亚砷酸盐处理对细胞进行应激，并再次测量形态、含RBM 45的包涵体的数量和细胞活力。我们还将检查细胞中RBM 45翻译后修饰的变化，与其他RNA结合蛋白的共定位，以及掺入应激颗粒以解决RBM 45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RBM 45基因靶点。接下来，我们将评估RBM 45对基因表达和选择性剪接调控的贡献。对于这些实验，RBM 45将通过shRNA从细胞中耗尽，并且基因表达和外显子剪接的变化将使用外显子连接敏感的微阵列来确定。在本提案的最后阶段，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中获得的结果与人类神经退行性疾病联系起来。将评价组织的RBM 45、内含物的脑区域特异性和细胞类型特异性表达，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RBM 45在正常细胞功能和疾病中作用的新信息。