TRANSLATION PROFILING AND NANOSCALE LOCALIZATION OF SYNAPTIC TRANSCRIPTS

描述（由申请人提供）：该资助的目的是开发用于突触mRNA转录物和蛋白质的细胞类型特异性鉴定和纳米级定位的第一组学水平方法。mRNA向突触位置的转运及其本地化翻译一直是一个有趣的现象，被认为对理解突触功能非常重要，但迄今为止仅使用少数选择系统进行了研究。最近的发现确定了翻译功能障碍在神经发育障碍中的关键作用，强调了全面了解突触翻译的重要性。我们提出联合收割机并聚焦于突触，两种最先进的方法，翻译核糖体亲和纯化（TRAP）和超分辨率随机光学重建显微镜（STORM）的新的识别和定位的突触转录本和本地翻译的蛋白质。TRAP的能力，使用eGFP标记的核糖体的细胞特异性表达，将允许我们特异性地从突触位点纯化翻译核糖体和相关mRNA，远离细胞体（Synap-TRAP）。捕获的mRNA的分析将为我们提供第一个无偏见的突触翻译蛋白质的全基因组分析。此外，将开发STORM以提供从Synap-TRAP中鉴定的RNA和蛋白质的纳米级定位，提供候选物的正交验证以及提供突触翻译的新超微结构信息。我们的综合方法可能会对我们理解学习和记忆以及特定突触病变中的正常突触功能产生重大影响。

DESCRIPTION (provided by applicant): The aim of this grant is to develop the first -omics level approach for the cell type specific identification and nanoscale localization of synaptic mRNA transcripts and proteins. The transport of mRNA to synaptic locations and their localized translation has long been an intriguing phenomenon, recognized to be of immense importance for understanding synaptic function, and yet hitherto studied using only a few select systems. Recent discoveries identifying a pivotal role for translation dysfunction in neurodevelopmental disorders underscore the importance of gaining a comprehensive understanding of synaptic translation. We propose to combine and focus at the synapse, two state-of-the-art methods, Translating Ribosome Affinity Purification (TRAP) and super-resolution STochastic Optical Reconstruction Microscopy (STORM) for the novel identification and localization of synaptic transcripts and locally translated proteins. The power of TRAP, using cell specific expression of eGFP tagged ribosomes, would allow us to specifically purify translating ribosomes and associated mRNA from synaptic sites, away from the cell body (Synap-TRAP). Analysis of captured mRNA would provide us with the first unbiased genome wide analysis of synaptically translated proteins. Further, STORM will be developed to provide nanoscale localization of RNA and proteins identified from Synap-TRAP, providing orthogonal validation of candidates as well as providing new ultrastructural information of synaptic translation. Our combined approaches are likely to have significant impact on our understanding of normal synaptic function in the context of learning and memory, as well as in specific synaptopathies.