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

 描述(申请人提供)：神经元调节它们何时何地合成新的蛋白质，以维持基础的和依赖活动的隔室的保真度。这一过程的控制在很大程度上是在翻译起始时调控的，翻译起始通常发生在适当的mRNA序列背景下的5‘-最大Aug密码子上。在简单模型系统中出现的数据表明，在所有转录本中，有很大一部分利用了未注释的上游开放阅读框架(UORF)。这些uORF通常在接近8月的密码子(与8月有一个碱基不同)时启动，并显示条件依赖的使用，这表明它们可能在翻译控制中发挥调节作用。这种非典型的启动事件是如何被调节的，它们扮演着什么角色 在神经元功能和疾病中的作用尚不清楚。这项研究的中心假设是，上游开放阅读框架的使用是神经元翻译动力学的重要调节因素，从未注释的uORF翻译产生的蛋白质将有助于神经元功能。我们的团队最近在神经退行性疾病脆性X相关颤动症共济失调综合征(FXTAS)中发现了一种与核苷酸重复相关的蛋白质翻译启动的新机制。FXTAS是FMR1基因5‘非翻译区CGG核苷酸重复扩增的结果。我们发现，这种重复扩增在5‘非编码区的一个接近8月的密码子处引发了起始，导致了通过 通过称为RAN(重复关联非AUG)转换的过程重复。这种翻译事件导致含有聚甘氨酸的蛋白质FMRPolyG的合成，即使在正常的重复大小下也是如此。这一提议将首先确定FMRPolyG在神经元中何时合成，既是基础的，也是对活动的反应。此外，将评估FMRPolyG合成对主要FMR1基因产物FMRP-a蛋白翻译的影响，FMRP-a蛋白与自闭症和智力残疾有关。为了确定类似的uORF利用事件在神经元中的流行程度，被称为核糖体图谱的新一代测序技术将被用于测量神经元转录组中5‘UTRs内的核糖体占有率。这些研究将利用细胞成像、生化和生物信息学技术来探索神经生物学中与突触可塑性和许多神经疾病相关的新领域。

 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.