探讨RNA代谢紊乱所致神经退行性疾病的发病机理

RNA metabolism abnormalities have been associated with disease, particularly neurodegenerative disorders. However, the mechanisms underlying disease are largely unknown, partially because of the absence of an ideal disease model that can directly link splicing abnormalities and neurodegeneration. Recently, we demonstrated that a mutation in one of the mouse multicopy U2 snRNA (small nuclear RNA) genes causes pre-mRNA splicing abnormalities and cerebellar neuron degeneration in a dosage-dependent manner. U2 snRNAs are basal components of the major spliceosome, which is responsible for the majority (99%) of human pre-mRNA splicing. Our transcriptome analysis revealed that only a subset of alternative splicing events, including splicing of small introns, is disrupted in the mutant cerebellum. Therefore, this mutant strain not only provides the definitive evidence to directly link the dysfunction of splicing to degeneration of neurons, but it also presents an experimental paradigm with which to better understand the disease mechanisms. The goal of this proposal is to study the disease mechanisms underlying neurodegeneration caused by RNA　metabolism abnormalities by using this novel mutant mouse strain and its disease-associated allele. In this application, we will determine the intronic regulatory elements essential for the retention of small introns, which are heavily retained in the mutant cerebellum, and examine whether the RNA transcripts containing intronic regulatory elements are toxic to neurons by sequstering the RNA binding proteins that recognize these regulatory elements. Second, we will identify a genetic modifier carried by BALB/cByJand Cast/EiJ, which can greatly attenuate the neurodegenerative phenotypes appearing in the mutant mouse brain. The data generated from these proposed experiments will not only deepen our understanding of the molecular and cellular pathways underlying pathogenesis of neurodegeneration caused by RNA metabolism abnomalites, but will also provide a basis for the future therapeutic development.

RNA代谢紊乱近年来被认为是神经退行性疾病的关键致病因子之一，但其如何致病一直是本研究领域急待解决的核心问题，部分因为缺少合适的动物模型。最近，我发现一个U2 snRNA基因上的突变，导致突变小鼠神经退行性病变 (Jia et al. 2012, Cell.)。U2 snRNA是参与前体mRNA剪接的基本元件，负责> 99％的人类前体mRNA剪接，但这一突变特异性影响选择性前体mRNA剪接，造成小内含子堆积。我们的发现首次为RNA代谢紊乱致神经退行性疾病提供了直接的证据。基于这一模型，我们提出异常地RNA序列堆积，通过竞争RNA结合蛋白，影响其正常功能而致病的假说。另外，我的最新结果提示，不同近交系小鼠存在显性遗传修饰因子，可大大缓解突变小鼠神经退行性病变。在此申请中，我们将验证异常RNA序列堆积的致病假说，并确定遗传修饰因子。我们的结果将对于理解RNA代谢紊乱的神经致病机制具有重要意义。

前体mRNA剪接对于基因功能调节和蛋白多样性有重要贡献，超过90%的人类基因有可变剪接；同时，大量的人类疾病与前体mRNA剪接或剪接调控紊乱相关。近年来越来越多的证据提示，前体mRNA剪接和相关的RNA代谢紊乱可能导致神经退行性疾病的发生发展。然而，异常代谢的RNA是如何被调控以及如何致病的机制不清。U2 snRNA (U2 small nuclear RNA)是参与前体mRNA 剪接的重要RNA成分，我们之前研究发现一个U2 snRNA基因突变小鼠伴有神经退行病变和上千个前体mRNA可变剪接紊乱。因此，这一基因突变和突变小鼠给我们提供了一个难得的机会，去认识异常代谢RNA的调控和致病的分子机制。通过小鼠正向遗传学筛选，我们发现可以缓解U2 snRNA突变引起的神经退行性病症的突变体小鼠，并成功鉴定到这个遗传修饰因子是一个编码核蛋白的基因。RNA-Seq结果显示，携带遗传修饰的小鼠还可以大大降低U2突变所致的前体mRNA剪接异常。利用免疫共沉淀－质谱分析方法，我们发现这个遗传修饰因子与剪接体复合物蛋白以及参与mRNA出核转运和降解的蛋白相互作用。我们的结果提示异常代谢的前体mRNA可能直接参与神经退行性疾病致病，我们找到的遗传修饰因子可能通过参与前体mRNA剪接和后续代谢发挥功能，这个遗传修饰因子未来有可能成为治疗神经退行性疾病的药物靶点。