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

描述(由申请人提供)：神经退行性疾病是导致发病率和死亡率的主要原因，预计此类疾病的负担在未来几十年将急剧增长。尽管如此，目前还没有有效的疗法来预防或治疗大多数神经退行性疾病。核苷酸重复序列的扩张是神经退行性变和神经系统疾病的常见原因，影响着全球数百万人。最近，核苷酸重复被发现促进了一种新的翻译起始形式，称为重复相关的非Aug(RAN)翻译，通过在没有Aug起始密码子的情况下发起翻译来产生含有同聚或二肽重复的蛋白质。我们的研究小组最近发现，RAN翻译与FMR1基因中CGG重复序列的扩张有关，这会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翻译从单独的开放阅读框中产生了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产品的产生有助于毒性，并在FXTAS患者脑组织中的神经元包裹体中积累，支持这一过程在FXTAS发病机制中的核心作用。然而，CGG重复究竟是如何引发RAN翻译的，以及这些过程需要通用翻译机制的哪些因素，目前尚不清楚。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，这取决于所利用的阅读框架。首先，我们认为扩展的CGG重复序列可以推迟FMR1 5‘UTR区的扫描预起始复合体(PIC)，从而允许在接近8月的起始密码子(如GUG)处启动以产生多甘氨酸蛋白。相反，我们认为CGG重复序列的扩张也可以直接招募翻译启动机制，并在重复序列本身内触发启动，从而产生多丙氨酸蛋白。为了验证这些翻译启动模型，我们将通过体外和基于细胞培养的实验来评估规范翻译启动的以下三个关键步骤的要求：1)5‘帽识别，)mRNA解旋和PIC扫描，以及3)开始密码子选择。目标1将确定每个阅读框架中的CGG RAN翻译是否依赖于帽。目的2将确定RNA解旋酶和重复不稳定蛋白的mRNA解离和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，我们将扩大这些研究，以确定调节这些相同的途径对CGG重复引起的果蝇毒性的影响。这些研究将确定RAN翻译允许在不同框架中扩大CGG重复的翻译启动的机制，同时为FXTAS和其他神经退行性重复扩张性疾病的发病机制和治疗发展提供关键的见解。

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.