Investigating the role of RNA exporter GLE1 in motor neuron disease

新出现的报告表明，在编码RNA加工蛋白和MND的基因突变之间存在着强烈的联系。mRNA加工在成人和儿童运动神经元疾病中的作用是该大学研究的主要焦点。GLE 1是一种mRNA输出因子，在胎儿运动神经元疾病致死性先天性挛缩综合征1型（LCCS 1）和肌萎缩侧索硬化症（ALS）病例中发生突变。然而，虽然在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，部分原因是关于RNA结合蛋白（包括Gle 1）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变性的研究工作对于这些破坏性疾病的治疗开发至关重要。然而，目前尚不清楚mRNA核输出的失调以及潜在的蛋白质合成改变是如何导致这种疾病的。我们假设运动神经元中RNA代谢和加工的破坏是导致与GLE 1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GLE 1。（ii）在细胞和iPS模型中鉴定具有改变的核输出的关键RNA分子（7-24个月）使用细胞质分级分离和人转录组阵列（基因芯片HTA2.0，Affyellow）：目的是鉴定在健康细胞中需要Gle 1核输出活性的所有mRNA分子和受Gle 1介导的神经变性影响的mRNA分子。首先使用Expression Console软件检查数据质量，并使用转录分析控制台程序（Affyoung）量化转录本，然后与量化CRAMA内不确定性的最先进工具进行比较。根据SEQC/MAQC-II联盟定义的质量标准，将进一步分析数据，以比较准确性和再现性。(iii)研究Gle 1疾病模型中mRNP复合物重构的改变。（24-36个月）：在这个目标下，我们计划在蛋白质组水平上鉴定mRNP的正常和潜在的疾病改变的Gle 1依赖性Dbp 5重塑。我们在mRNP捕获检测方面拥有丰富的经验，可在活细胞UV交联后特异性纯化和鉴定直接与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.