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

CGG RAN 翻译在脆性 X 相关震颤/共济失调综合征中的机制

• 批准号: 8780143
• 负责人: Michael G Kearse
• 金额: $ 5.23万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8780143
• 关键词: 5' Untranslated Regions; Address; Affect; Alanine; Binding; Biochemical; Biological Models; Brain; C9ORF72; CGG repeat; CGG repeat expansion; Cell Culture Techniques; Cells; Codon Nucleotides; Complex; Data; Degenerative Disorder; Dipeptides; Disease; Dominant-Negative Mutation; Drosophila genus; FMR1; FMR1 Gene; FXTAS; Fellowship; Gene Expression Regulation; Gene Mutation; Glycine; Homo; In Vitro; Inherited; Initiator Codon; Internal Ribosome Entry Site; Messenger RNA; Modeling; Morbidity - disease rate; Nerve Degeneration; Neurodegenerative Disorders; Neurologic; Neurons; Nuclear Inclusion; Nucleotides; Open Reading Frames; Pathogenesis; Pathway interactions; Patients; Peptide Initiation Factors; Preparation; Prevention; Process; Production; Proteins; RNA Helicase; Reading Frames; Recruitment Activity; Research Personnel; Research Project Grants; Role; Scanning; Structure; Testing; Therapeutic; Toxic effect; Training; Translation Initiation; Translations; Ubiquitin; United States; Validation; Work; age related; base; brain tissue; career; helicase; insight; mortality; nervous system disorder; new therapeutic target; novel; public health relevance; research study; therapeutic development

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.

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，预计此类疾病的负担在未来几十年内将急剧增加。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。核苷酸重复序列扩增是神经变性和神经系统疾病的常见原因，影响着全球数百万人。最近，已经发现核苷酸重复序列促进一种新形式的翻译起始，称为重复序列相关非AUG（RAN）翻译，通过在AUG起始密码子不存在的情况下启动翻译产生含有同源聚合物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FMR 1基因内的CGG重复扩增相关，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翻译从单独的开放阅读框产生含有聚甘氨酸和聚丙氨酸的蛋白质。聚甘氨酸RAN产物的产生在简单模型系统中导致毒性，并在FXTAS患者脑组织中的神经元内含物中积累，支持该过程在FXTAS发病机制中的中心作用。然而，CGG重复序列如何确切地引起RAN翻译以及这些过程所需的一般翻译机制的哪些因素目前尚不清楚。基于我们的初步数据，我们假设CGG RAN翻译可以通过依赖于所利用的阅读框架的至少两种独立的新机制发生。首先，我们提出扩展的CGG重复序列可以停止扫描FMR 1的5 'UTR中的前起始复合物（PIC），从而允许在接近AUG的起始密码子（如GUG）处起始以产生聚甘氨酸蛋白。相反，我们认为CGG重复序列的扩增也可以直接募集翻译起始机制，并在重复序列本身内引发起始，从而产生聚丙氨酸蛋白。为了测试这些翻译起始模型，我们将通过体外和基于细胞培养的实验来评估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）起始密码子选择。目标1将确定每个阅读帧中的CGG RAN翻译是否是帽依赖性的。目的2将确定RNA解旋酶和重复不稳定蛋白的mRNA解旋和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翻译允许在扩展的CGG重复序列的不同框架中进行翻译起始的机制，同时为FXTAS和其他神经退行性重复序列扩展疾病的发病机制和治疗开发提供重要见解。