Mechanisms of CGG RAN translation in Fragile X-associated tremor/ataxia syndrome

描述（由申请人提供）：神经退行性疾病是发病率和死亡率的主要原因，预计在未来几十年，此类疾病的负担将急剧增长。尽管如此，目前还没有有效的治疗方法来预防或治疗大多数神经退行性疾病。核苷酸重复扩增是神经变性和神经系统疾病的常见原因，影响着全世界数百万人。最近，人们发现核苷酸重复序列可以促进一种新的翻译起始形式，即重复相关的非AUG （RAN）翻译，通过在没有AUG起始密码子的情况下启动翻译，产生含有同聚或二肽重复序列的蛋白质。我们的研究小组最近发现RAN翻译与FMR1基因内CGG重复扩增有关，导致神经退行性疾病脆性x相关震颤/共济失调综合征（FXTAS）。我们的初步数据表明，CGG RAN翻译产生了来自不同开放阅读框的含有聚甘氨酸和聚丙氨酸的蛋白质。在简单的模型系统中，聚甘氨酸RAN产物的产生有助于毒性，并在FXTAS患者脑组织中的神经元包涵体中积累，支持该过程在FXTAS发病机制中的核心作用。然而，目前尚不清楚CGG重复序列究竟如何引发RAN翻译，以及这些过程需要一般翻译机制中的哪些因素。根据我们的初步数据，我们假设CGG - RAN翻译可以通过至少两种独立的新机制发生，这些机制依赖于所使用的阅读框。首先，我们提出扩增的CGG重复序列可以阻止FMR1 5'UTR中的扫描起始前复合物（PICs），从而允许在接近aug的起始密码子（如GUG）起始以产生聚甘氨酸蛋白。相比之下，我们提出CGG重复扩增也可以直接招募翻译起始机制并触发重复本身的起始以产生聚丙氨酸蛋白。为了验证这些翻译起始模型，我们将通过体外实验和基于细胞培养的实验来评估标准翻译起始的以下三个关键步骤的要求：1)5'帽识别，)mRNA解绕和PIC扫描，以及3)开始密码子选择。目标1将确定每个阅读帧中的CGG RAN翻译是否依赖于上限。目的2将确定RNA解旋酶和重复不稳定蛋白的mRNA解绕和PIC扫描是否影响CGG RAN翻译。目的3将确定起始因子在非aug起始密码子选择中的作用。然后，我们将扩展这些研究，以确定调节这些相同的途径对果蝇中CGG重复引起的毒性的影响。这些研究将确定RAN翻译在扩展的CGG重复序列的不同框架中允许翻译起始的机制，同时为FXTAS和其他神经退行性重复扩增疾病的发病机制和治疗发展提供重要见解。

DESCRIPTION (provided by applicant): Neurodegenerative disorders are a leading cause of morbidity and mortality, and the burden of such disorders is expected to grow dramatically over the coming decades. Despite this, there is currently no effective therapeutics for prevention or treatment of most neurodegenerative conditions. Nucleotide repeat expansions are a common cause of neurodegeneration and neurological illness, affecting millions of people worldwide. Recently, nucleotide repeats have been found to promote a novel form of translational initiation known as Repeat-Associated Non-AUG (RAN) translation, producing homo-polymeric or dipeptide repeat containing proteins by initiating translation in the absence of an AUG start codon. Our group has recently discovered that RAN translation occurs in association with CGG repeat expansions within the FMR1 gene that cause the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). Our preliminary data demonstrates that CGG RAN translation produces both poly-glycine and poly-alanine containing proteins from separate open reading frames. Production of the poly-glycine RAN product contributes to toxicity in simple model systems and accumulates in neuronal inclusions in FXTAS patient brain tissue, supporting a central role for this process in FXTAS pathogenesis. However, how exactly CGG repeats elicit RAN translation and what factors of the general translation machinery are required for these processes is currently unknown. Based on our preliminary data, we hypothesize that CGG RAN translation can occur by at least two separate novel mechanisms that are dependent on the utilized reading frame. First, we propose that expanded CGG repeats can stall scanning pre-initiation complexes (PICs) in the 5'UTR of FMR1, allowing for initiation at a near-AUG start codon (such as GUG) to produce the poly-glycine protein. In contrast, we propose that CGG repeat expansions can also directly recruit translational initiation machinery and trigger initiatin within the repeat itself to produce the poly-alanine protein. To test these translational initiatio models, we will assess the requirements for the following three key steps of canonical translation initiation through in vitro and cell culture-based experiments: 1) 5' cap recognition, ) mRNA unwinding and PIC scanning, and 3) start codon selection. Aim 1 will determine if CGG RAN translation in each reading frame is cap-dependent. Aim 2 will determine if mRNA unwinding and PIC scanning by RNA helicases and repeat-destabilizing proteins influence CGG RAN translation. Aim 3 will determine the role of initiation factors in non-AUG start codon selection. We will then extend these studies to determine the impact of modulating these same pathways has on CGG repeat elicited toxicity in Drosophila. These studies will identify the mechanisms by which RAN translation allows for translational initiation in different frames at expanded CGG repeats, while providing critical insights into pathogenesis and therapeutic development for FXTAS and other neurodegenerative repeat expansion disorders.