Identifying the molecular mechanisms of GEMIN5 mutations in a novel cerebellar ataxia syndrome

摘要 GEMIN 5是一种RNA结合蛋白，对生存运动神经元（SMN）的组装至关重要 复杂. GEMIN 5促进小核核糖核蛋白（snRNP; 剪接体的结构单元）。我们发现了新的常染色体隐性变异， GEMIN 5基因在多例运动功能障碍、共济失调和小脑性共济失调患者中的表达 萎缩 我们的研究旨在了解突变型GEMIN 5的分子机制， 导致我们病人神经系统异常的原因我们发现病人a 患者iPSC中GEMIN 5蛋白水平显著降低和蛋白稳定性降低 神经元提示可能的功能丧失机制。我们的体外组装试验 显示GEMIN 5变体干扰snRNP组装形成。了解 GEMIN 5功能丧失的后果，我们敲低内源性尸僵， 人GEMIN 5的果蝇同源物，在果蝇中。尸僵击倒（钻机） 导致运动功能障碍，寿命缩短和发育迟缓。有趣的是，我们 观察到CoQ 10水平在人类患者细胞中显著降低， 果蝇模型。辅酶Q10治疗减少了人类GEMIN 5的病程 患者我们使用CRISPR/cas9生成了Gemin 5的小鼠模型， 对小鼠我们的研究旨在了解GEMIN 5的分子机制 通过1）进行突变型GEMIN 5患者神经元的功能分析; 2）检查是否 GEMIN 5变体导致体内线粒体功能障碍和iPSC神经元;以及3） 在小鼠模型中研究GEMIN 5突变的机制。我们希望能找出 在人类患者中受到干扰的分子途径。

Abstract GEMIN5, an RNA-binding protein, is essential for assembly of the Survival Motor Neuron (SMN) complex. GEMIN5 facilitates the formation of small nuclear ribonucleoproteins (snRNPs; the building blocks of spliceosomes). We identified novel autosomal recessive variants in the GEMIN5 gene in multiple patients presenting with motor dysfunction, ataxia, and cerebellar atrophy. Our proposed studies are aimed to understand the molecular mechanisms of mutant GEMIN5 responsible for causing the neurological abnormalities in our patients. We found that patient a significant decrease in GEMIN5 protein levels and reduced protein stability in patient iPSC neurons suggesting a possible loss of function mechanism. Our in vitro assembly assay showed that GEMIN5 variants perturb snRNP assembly formation. To understand the consequences of loss of function GEMIN5, we knockdown endogenous rigor mortis, the Drosophila homologue of human GEMIN5, in Drosophila. Knockdown of rigor mortis (rig) caused motor dysfunction, reduced life span and developmental delay. Interestingly, we observed that CoQ10 levels were significantly reduced in human patient cells and our drosophila model. Treatment with CoQ10 reduced the disease course in human GEMIN5 patients. We generated a mouse model of Gemin5 using CRISPR/cas9 and found early lethality in mice. Our proposed studies are aimed to understand the molecular mechanisms of GEMIN5 by 1) conducting functional analysis of mutant GEMIN5 patient neurons; 2) examining if GEMIN5 variants cause mitochondrial dysfunctions in vivo and iPSC neurons; and 3) investigating the mechanisms of GEMIN5 mutations in mouse models. We expect to identify the molecular pathways that are perturbed in human patients.