TRANSLATION PROFILING AND NANOSCALE LOCALIZATION OF SYNAPTIC TRANSCRIPTS

描述(由申请人提供)：这笔赠款的目的是开发一种第一级组学方法，用于突触mRNA转录本和蛋白质的细胞类型特定鉴定和纳米级定位。长期以来，信使核糖核酸到突触位置的运输及其局部化翻译一直是一个有趣的现象，被认为对理解突触功能具有极其重要的意义，但到目前为止，只有几个精选的系统进行了研究。最近发现翻译障碍在神经发育障碍中起着关键作用，这突显了全面了解突触翻译的重要性。我们建议结合并专注于突触这两种最先进的方法，即翻译核糖体亲和力纯化(TRAP)和超分辨率随机光学重建显微镜(STORM)，用于突触转录本和局部翻译蛋白的新识别和定位。TRAP的能力，利用EGFP标记的核糖体的细胞特异性表达，将使我们能够特异性地从突触部位提纯翻译的核糖体和相关的mRNA，远离细胞体(Synap-Trap)。对捕获的mRNA的分析将为我们提供第一个无偏倚的全基因组范围的突触翻译蛋白分析。此外，STORM将被开发来提供从Synap-Trap中识别的RNA和蛋白质的纳米级定位，提供候选的正交验证以及提供新的突触翻译的超微结构信息。我们的结合方法可能会对我们在学习和记忆背景下以及特定突触病变中对正常突触功能的理解产生重大影响。

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.