The toxicity of the RNA CGG repeats in FXTAS

描述(申请人提供)：脆性X相关震颤/共济失调综合征(FXTAS)是一种进行性神经退行性疾病，以震颤伴共济失调、脑萎缩和认知缺陷为特征。FXTAS是由FMR1基因5‘非编码区的CGG扩增引起的，其长度从~55到200个重复(CGG55-200)不等。同一基因中超过200个重复的CGG扩增会导致脆性X综合征(FXS)，这是一种与FXTAS不同的智力残疾。长CGG扩增抑制FMR1基因的转录，而短扩增则上调FMR1的转录。大量模型表明，FMR1基因外含有CGG55-200重复序列或RNA CGG55-200重复序列的FMR1基因可导致神经退行性变。FXTAS的主要病理特征是形成泛素(Ub)阳性包涵体，其中包含蛋白酶体和几种RNA结合蛋白。然而，Ub-蛋白酶体系统和RNA结合蛋白在FXTAS病理中的作用尚不清楚。RNA CGG重复序列的主要靶点也仍然未知。我实验室之前的工作集中在由RNA、CUG和CCUG重复引起的1型和2型强直性肌营养不良的机制上。在这些研究过程中，我们发现CUG和CCUG重复序列通过与不同的RNA结合蛋白相互作用以及通过形成可溶的和沉淀的RNA-蛋白质复合体(RPC)来改变RNA的代谢。CuG和CCUG RNA与RNA结合蛋白CUGBP1形成可溶性络合物，增加CUGBP1的稳定性。CCUG重复序列还通过形成大的RPC来影响蛋白质的降解，RPC包含蛋白酶体和与压力相关的蛋白质。我们认为，与DM类似，短(55-200)RNA CGG重复形成可溶的和沉淀的RPC。我们认为可溶性rCGG-RPC中可能含有CUGBP1、PUR�和hnRNPA2/B1等核糖核酸结合蛋白，影响其功能。为了支持这一假说，我们发现FMR1-CGG98敲击小鼠的脑中CUGBP1表达增加。已知的是，沉淀的rCGG重复序列(焦点)与Sam68、MBNL1和hnRNP G结合。我们认为，这些蛋白与沉淀的RPC的过度结合可能吸引了蛋白酶体，导致Ub阳性包涵体。这一应用的主要假设是，FXTAS的脑萎缩和神经变性是由于：(1)可溶和沉淀的RPC结合的RNA结合蛋白对RNA加工的错误调节；以及(2)由于蛋白质降解的改变。为了确定这些毒性事件在FXTAS病理中的作用，我们建议使用TET调节的细胞模型(目标1)确定rCGG100和FMR1-CGG90 RNA的主要靶点。在不同年龄小鼠的rCGG90转基因和FMR1-CGG98敲击中，将确定CUGBP1的升高在体内神经退行性变中的作用(目标2)。这些研究将有助于明确FXTAS的分子机制，并为FXTAS治疗的发展提供背景。

DESCRIPTION (provided by applicant): Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a progressive neurodegenerative disease characterized by tremor with ataxia, brain atrophy and cognitive defects. FXTAS is caused by CGG expansions, varying in the length from ~55 to 200 repeats (CGG55-200), in the 5' UTR of the FMR1 gene. The CGG expansions more than 200 repeats in the same gene cause the Fragile X Syndrome (FXS), an intellectual disability, distinct from FXTAS. Long CGG expansions silence the transcription of the FMR1 gene; while short expansions elevate the transcription of the FMR1. Numerous models show that the FMR1 mRNA with CGG55-200 repeats or RNA CGG55-200 repeats outside of the FMR1 mRNA cause neurodegeneration. The main pathologic feature of FXTAS is the formation of the ubiquitin (Ub)-positive inclusions that contain the proteasome and several RNA-binding proteins. However, the role of the Ub- proteasome system and RNA-binding proteins in the FXTAS pathology is not understood. The primary targets of the RNA CGG repeats also remain unknown. Previous work in my lab was focused on the mechanisms for Myotonic Dystrophies type 1 and type 2 which are caused by RNA CUG and CCUG repeats. In the course of these studies, we have found that CUG and CCUG repeats alter RNA metabolism by interaction with different RNA-binding proteins and by formation of soluble and precipitated RNA-protein complexes (RPCs). CUG and CCUG RNAs form soluble complexes with RNA-binding protein, CUGBP1, increasing CUGBP1 stability. CCUG repeats also affect protein degradation via formation of large RPCs, containing the proteasome and stress-related proteins. We propose that, similar to DM, short (55-200) RNA CGG repeats form soluble and precipitated RPCs. We suggest that soluble rCGG-RPCs might contain RNA-binding proteins such as CUGBP1, Pur� and hnRNP A2/B1, affecting their function. In support of this hypothesis, we have found that CUGBP1 is increased in brains of FMR1-CGG98 knock in mice. It is known that the precipitated rCGG repeats (foci) bind Sam68, MBNL1 and hnRNP G. We suggest that the excessive binding of these proteins to the precipitated RPCs may attract the proteasome leading to the Ub-positive inclusions. The main hypothesis of this application is that the brain atrophy and neurodegeneration in FXTAS is due to: (1) misregulation of RNA processing by RNA-binding proteins bound in soluble and precipitated RPCs; and (2) due to altered protein degradation. To determine the role of these toxic events in the FXTAS pathology, we propose to identify the primary targets of the rCGG100 and the FMR1-CGG90 RNAs using a tet-regulated cell model (Aim 1). The role of elevation of CUGBP1 in the neurodegeneration in vivo will be determined in rCGG90 transgenic and FMR1-CGG98 knock in mice of different age (Aim 2). These studies will help to identify the molecular mechanism of FXTAS and will provide a background for the development of therapy for FXTAS.