Sequestration of multiple RNA recognition motif-containing proteins by C9orf72 repeat expansions.
Sequestration of multiple RNA recognition motif-containing proteins by C9orf72 repeat expansions.
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DOI：
10.1093/brain/awu120
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发表时间：
2014-07
期刊：
影响因子：
--
通讯作者：
Shaw PJ
中科院分区：
文献类型：
--
作者：
Cooper-Knock J;Walsh MJ;Higginbottom A;Robin Highley J;Dickman MJ;Edbauer D;Ince PG;Wharton SB;Wilson SA;Kirby J;Hautbergue GM;Shaw PJ
C9orf72基因中GGGGCC重复序列的扩展导致家族性肌萎缩侧索硬化症(ALS)和额颞部痴呆，但其潜在机制尚不清楚。使用RNA下拉和免疫组织化学在肌萎缩侧索硬化症生物样本，库珀-敲击等人。识别与重复扩增相结合的蛋白质。RNA剪接中断和/或核输出可能是C9orf72-ALS发病的基础。C9orf72基因的GGGGCC重复扩增是肌萎缩侧索硬化症和额颞部退行性变最常见的遗传变异，但发病机制尚不清楚。最近的报道表明，转录的重复序列可能形成有毒的RNA焦点，隔离各种RNA加工蛋白。对于结合伙伴的身份缺乏共识，需要对整个神经元蛋白质组进行研究。应用核糖核酸荧光原位杂交技术，首次在肌萎缩侧索硬化症C9orf72重复扩增(C9orf72+)患者外周和中枢神经系统标本中发现了核质RNA灶，但未发现C9orf72重复扩增(C9orf72−)患者和正常对照组。此外，在所研究的病例中，病灶阳性神经元的分布与临床表型相关(t检验P<；0.05)。正如预期的那样，RNA焦点被核糖核酸酶处理消融。有趣的是，我们在一例无症状的C9orf72+携带者的成纤维细胞中发现了病灶。接下来，我们用GGGGCC5进行了下拉分析，并结合质谱分析，确定了GGGGCC重复扩张的候选结合伙伴。含有RNA识别基序并参与剪接、信使RNA核输出和/或翻译的蛋白质显著丰富。在C9orf72+肌萎缩侧索硬化症患者的中枢神经系统组织中，免疫组织化学显示RNA灶与SRSF2、hnRNP H1/F、ALYREF和hnRNP A1共存于小脑颗粒细胞，与SRSF2、hnRNP H1/F和ALYREF共存于运动神经元，是肌萎缩侧索硬化症的主要病理靶点。体外紫外光交联法证实蛋白质与GGGGCC重复RNA直接结合。共定位只在一小部分RNA焦点中检测到，这表明动态隔离而不是不可逆转的结合。免疫组织化学结果显示，有或无核糖核酸灶的神经元同样可能出现TdP-43核耗竭(χ2 P=0.75)或多聚GA二肽重复蛋白包涵体(χ2 P=0.4 6)。我们的发现提出了两种非排他性的致病机制：(I)RNA加工蛋白的功能耗尽导致信使RNA剪接的中断；(Ii)扩增的C9orf72前信使RNA通过与信使RNA输出适配蛋白(S)不适当的结合而被许可用于核出口，导致细胞质重复相关的非ATG翻译和潜在有毒的二肽重复蛋白的形成。
Expansion of GGGGCC repeats in C9orf72 causes familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but the underlying mechanism is unclear. Using RNA pulldown and immunohistochemistry in ALS biosamples, Cooper-Knock et al. identify proteins that bind to the repeat expansions. Disrupted RNA splicing and/or nuclear export may underlie C9orf72-ALS pathogenesis. GGGGCC repeat expansions of C9orf72 represent the most common genetic variant of amyotrophic lateral sclerosis and frontotemporal degeneration, but the mechanism of pathogenesis is unclear. Recent reports have suggested that the transcribed repeat might form toxic RNA foci that sequester various RNA processing proteins. Consensus as to the identity of the binding partners is missing and whole neuronal proteome investigation is needed. Using RNA fluorescence in situ hybridization we first identified nuclear and cytoplasmic RNA foci in peripheral and central nervous system biosamples from patients with amyotrophic lateral sclerosis with a repeat expansion of C9orf72 (C9orf72+), but not from those patients without a repeat expansion of C9orf72 (C9orf72−) or control subjects. Moreover, in the cases examined, the distribution of foci-positive neurons correlated with the clinical phenotype (t-test P < 0.05). As expected, RNA foci are ablated by RNase treatment. Interestingly, we identified foci in fibroblasts from an asymptomatic C9orf72+ carrier. We next performed pulldown assays, with GGGGCC5, in conjunction with mass spectrometry analysis, to identify candidate binding partners of the GGGGCC repeat expansion. Proteins containing RNA recognition motifs and involved in splicing, messenger RNA nuclear export and/or translation were significantly enriched. Immunohistochemistry in central nervous system tissue from C9orf72+ patients with amyotrophic lateral sclerosis demonstrated co-localization of RNA foci with SRSF2, hnRNP H1/F, ALYREF and hnRNP A1 in cerebellar granule cells and with SRSF2, hnRNP H1/F and ALYREF in motor neurons, the primary target of pathology in amyotrophic lateral sclerosis. Direct binding of proteins to GGGGCC repeat RNA was confirmed in vitro by ultraviolet-crosslinking assays. Co-localization was only detected in a small proportion of RNA foci, suggesting dynamic sequestration rather than irreversible binding. Additional immunohistochemistry demonstrated that neurons with and without RNA foci were equally likely to show nuclear depletion of TDP-43 (χ2 P = 0.75) or poly-GA dipeptide repeat protein inclusions (χ2 P = 0.46). Our findings suggest two non-exclusive pathogenic mechanisms: (i) functional depletion of RNA-processing proteins resulting in disruption of messenger RNA splicing; and (ii) licensing of expanded C9orf72 pre-messenger RNA for nuclear export by inappropriate association with messenger RNA export adaptor protein(s) leading to cytoplasmic repeat associated non-ATG translation and formation of potentially toxic dipeptide repeat protein.
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