The Functional Role of RBM45 in Gene Expression and Neurodegeneration

描述（由申请人提供）：肌萎缩性侧索硬化症（ALS）是一种进行性，不可避免的致命神经退行性疾病。病理上，ALS的特点是皮质脊髓束运动神经元的死亡，导致许多衰弱症状，最终导致瘫痪和死亡。对肌萎缩侧索硬化症神经退行性变的分子机制的研究表明，细胞死亡中RNA加工和RNA结合蛋白的胞浆内聚集受损。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RBM45作为假定的疾病生物标志物。通过免疫组化对ALS患者脊髓组织RBM45的进一步研究发现，运动神经元中的RBM45阳性胞浆内包涵体与含有其他ALS相关RNA结合蛋白TDP-43和FUS的包涵体外观相似。我们现在试图通过研究RBM45的正常功能及其对神经退行性疾病病理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、包涵体的形成和活力。我们还将过度表达缺乏假定的核定位信号的蛋白质的自然存在的异构体，以确定这种变体在体外聚集的倾向。该提案的下一个目标是了解RBM45在细胞应激反应中的作用（如果有的话）。将通过亚砷酸盐处理细胞，并再次测量细胞形态、含rbm45内含物的数量和细胞活力。我们还将检测细胞中RBM45翻译后修饰的变化，与其他RNA结合蛋白的共定位，以及与应激颗粒的结合，以解决RBM45在应激期间的功能。在该提案的第二阶段，RBM45基因靶点将通过UV交联和免疫沉淀结合RNA-Seq来确定。接下来，我们将评估RBM45对基因表达和选择性剪接调控的贡献。在这些实验中，RBM45将通过shRNA从细胞中去除，基因表达和外显子剪接的变化将使用外显子连接敏感微阵列来确定。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，将目的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，这些实验将提供关于RBM45在正常细胞功能和疾病中的作用的新信息。

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.