Investigating the role of RNA exporter GLE1 in motor neuron disease

新出现的报告表明，编码rna加工蛋白的基因突变与mnd之间存在着密切的联系。mRNA加工在成人和儿童形式的运动神经元疾病中的作用是该大学研究的主要焦点。GLE1是一种mRNA输出因子，在胎儿运动神经元障碍致死性先天性挛缩综合征1型（LCCS1）和肌萎缩侧索硬化症（ALS）病例中发生突变。然而，尽管该领域已经取得了重大的遗传发现，但ALS患者目前没有治疗选择，部分原因是包括Gle1在内的rna结合蛋白缺乏如何导致ALS的关键问题仍未得到解答。因此，旨在了解这些基因突变如何导致神经退行性变的研究工作对于促进这些破坏性疾病的治疗发展至关重要。有确凿的证据表明Gle1与神经变性有关。然而，目前尚不清楚mRNA核输出失调和潜在的蛋白质合成改变是如何导致疾病的。我们假设运动神经元中RNA代谢和加工的破坏是导致与GLE1基因突变相关的ALS的关键事件。因此，我们的研究计划和目标是：(i)设计CRISPR- Cas9来调节运动神经元和神经胶质细胞中的Gle1（1-7个月）；（ii）使用细胞质分离和人类转录组阵列（GeneChip HTA2.0, Affymetrix）鉴定细胞和iPS模型中核输出改变的关键RNA分子（7-24个月）：目的是鉴定健康细胞和受Gle1介导的神经退行性疾病影响的细胞中所有需要Gle1核输出活性的mRNA分子。首先使用Expression Console软件检查数据质量，然后使用转录分析控制台程序（Affymetrix）对转录进行量化，然后使用最先进的工具对PUMA内的不确定性进行量化。根据SEQC/MAQC-II联盟定义的质量标准，将进一步分析数据以比较准确性和可重复性。（iii）研究Gle1疾病模型中mRNP复合体重塑的改变。（24-36个月）：在此目标下，我们计划在蛋白质组水平上识别正常和潜在疾病改变的gl1依赖性dbp5 - mRNPs重塑。我们在mRNP捕获分析方面有丰富的经验，可以在活细胞紫外线交联后纯化和鉴定直接与PolyA+ RNA结合的蛋白质。

Emerging reports indicate that there is a strong link between mutations in genes encoding RNA-processing proteins and MNDs. The role of mRNA processing in adult and childhood forms of motor neuron disease is a major focus of research in the University. GLE1 is an mRNA export factor that is mutated in the fetal motor neuron disorder lethal congenital contracture syndrome type 1 (LCCS1) and in amyotrophic lateral sclerosis (ALS) cases. However, while significant genetic discoveries have been made in the field, patients with ALS currently have no treatment options, in part because key questions on how deficiency in RNA-binding proteins, including Gle1, causes ALS remain unanswered. Thus, research efforts aimed at understanding how mutations in these genes cause neurodegeneration are of the utmost importance to enable therapeutic development for these devastating disorders.There is solid evidence linking Gle1 to neurodegeneration. It is however unclear how dysregulation of mRNA nuclear export, and potentially altered protein synthesis, cause the disease. We hypothesize that disruption of RNA metabolism and processing in motor neurons is a key event leading to ALS linked to mutations in the GLE1 gene.Our research plan and objectives are therefore to:(i) Design CRISPR- Cas9 to modulate Gle1 in motor neurons and glia cells (1-7 months)(ii) Identify key RNA molecules with altered nuclear export in cell and iPS models (7-24 month) using cytoplasmic fractionation and human transcriptome arrays (GeneChip HTA2.0, Affymetrix): The aim is to identify all mRNA molecules that require the nuclear export activity of Gle1 in healthy cells and those affected in Gle1-mediated neurodegeneration. Data quality will initially be checked using the Expression Console software and transcripts quantified with the Transcription Analysis Console program (Affymetrix) prior to comparison with state-of-the-art tools that quantify uncertainty within PUMA. Data will further be analysed to compare accuracy as well as reproducibility according to the quality standards defined by the SEQC/MAQC-II Consortium.(iii) Investigate the alteration of mRNP complex remodeling in disease models of Gle1. (24-36 months): under this aim, we plan to identify normal and potentially disease-altered Gle1-dependent Dbp5-remodelling of mRNPs at the proteome level. We have extensive experience with mRNP capture assays that specifically allow purification and identification of proteins directly bound to PolyA+ RNA following UV-cross-linking of live cells.