Upstream open reading frames in neuronal function: a singular and genome-wide approach

 描述（由申请人提供）：神经元调节何时何地合成新蛋白质，以维持基础和活性依赖性房室保真度。这一过程的控制在翻译起始时受到很大程度的调节，翻译起始通常发生在适当的mRNA序列背景下的最5 '端AUG密码子处。简单模型系统中的新兴数据表明，未注释的上游开放阅读框架（uORF）在所有转录本中的很大一部分上被利用。这些uORF通常起始于接近AUG（与AUG相差一个碱基）的密码子，并显示条件依赖性使用，表明它们可能在翻译控制中起调节作用。这些非典型启动事件是如何被介导的，它们在其中扮演什么样的角色？ 在神经元功能和疾病中的作用是未知的。这项资助的中心假设是上游开放阅读框架的使用是神经元中翻译动力学的重要调节因子，并且通过从未注释的uORF翻译产生的蛋白质将有助于神经元功能。我们的研究小组最近发现了一种新的机制，蛋白质翻译起始与核苷酸重复在神经退行性疾病脆性X相关震颤共济失调综合征（FXTAS）。FXTAS由FMR 1基因的5'非翻译区（UTR）中的CGG核苷酸重复扩增产生。我们发现，这种重复扩增在5 'UTR中的近AUG密码子处启动，导致通过 通过称为RAN（重复相关非AUG）翻译的过程重复。该翻译事件导致合成含多聚甘氨酸的蛋白质，FMRpolyG，即使在正常的重复序列大小下。该提案将首先确定何时在神经元中合成FMRpolyG，包括基础合成和对活动的响应。此外，将评估FMRpolyG合成对主要FMR 1基因产物FMRP--一种与自闭症和智力残疾有关的蛋白质--翻译的影响。为了确定神经元中类似uORF利用事件的普遍性，将使用称为核糖体谱分析的新的下一代测序技术来测量神经元转录组中5 'UTR内的核糖体占有率。这些研究将采用细胞成像、生物化学和生物信息学技术，探索神经生物学中与突触可塑性以及许多神经系统疾病相关的新领域。

 DESCRIPTION (provided by applicant): Neurons regulate when and where they synthesize new proteins to maintain basal and activity-dependent compartmental fidelity. Control of this process is largely regulated at translational initiation which typically occurs at the 5'-most AUG codon in an appropriate mRNA sequence context. Emerging data in simple model systems suggests that un-annotated, upstream open reading frames (uORFs) are utilized on a significant fraction of all transcripts. These uORFs often initiate at near-AUG (one base different from AUG) codons and display condition-dependent usage, suggesting they may play a regulatory role in translational control. How such atypical initiation events are mediated and what roles they play in neuronal function and disease are unknown. The central hypothesis of this grant is that upstream open reading frame usage is an important regulator of translational dynamics in neurons, and that proteins produced by translation from un-annotated uORFs will contribute to neuronal function. Our group recently discovered a novel mechanism for protein translational initiation associated with nucleotide repeats in the neurodegenerative disorder Fragile X-associated Tremor Ataxia Syndrome (FXTAS). FXTAS results from a CGG nucleotide repeat expansion in the 5' untranslated region (UTR) of the FMR1 gene. We discovered that this repeat expansion elicits initiation at a near-AUG codon in the 5'UTR, resulting in translation through the repeat via a process known as RAN (Repeat Associated Non-AUG) translation. This translation event leads to synthesis of a poly-glycine containing protein, FMRpolyG, even at normal repeat sizes. This proposal will first determine when FMRpolyG is synthesized in neurons both basally and in response to activity. Further, the impact of FMRpolyG synthesis on translation of the major FMR1 gene product, FMRP- a protein implicated in autism and intellectual disability will be assessed. To determine the prevalence of similar uORF utilization events in neurons, the novel next-generation sequencing technique known as ribosomal profiling will be used to measure ribosomal occupancy within 5'UTRs across the neuronal transcriptome. These studies will employ cellular imaging, biochemical and bioinformatics techniques to explore a new area in neurobiology with relevance to both synaptic plasticity as well as numerous neurological diseases.