Tools for genome-wide profiling of mRNA translated in in-vivo dendrites

描述（由申请人提供）：大脑通过不断调整神经元之间的连接强度来处理和存储信息。为了调节这些被称为突触的连接的强度，神经元必须合成新的蛋白质。蛋白质可以在神经元的胞体中合成，然后通过树突到达突触。另外，蛋白质也可以在靠近突触的树突内合成。这种树突蛋白的合成提供了一种快速有效的方法来改变单个突触连接的强度。树突蛋白的合成对正常的大脑发育和认知功能至关重要。当树突蛋白合成受损时，例如脆性X综合征，就会导致严重的神经发育和认知缺陷。尽管树突状蛋白的合成很重要，但人们对它的了解并不完全。剩下的一个主要问题是：哪些蛋白质可以在树突中合成？这个项目将测试和应用新的工具来回答这个问题。该工具允许从体内树突中选择性分离核糖体结合的信使RNA （mRNA）。由于核糖体结合mRNA通过mRNA翻译过程合成蛋白质，因此核糖体结合的翻译mRNA直接反映了合成的蛋白质。为了完全表征树突翻译mRNA，该项目将使用下一代测序技术对样品中的每个mRNA分子进行测序（RNA-Seq）。了解每个树突翻译mRNA分子的序列不仅回答了哪些蛋白质可以在树突中合成的问题，而且还提供了对合成的特定蛋白质同种异构体的见解。该项目生成的序列数据也可用于发现树突状mRNA群共享的RNA基序。其中一些基序可以调节mRNA在树突内的转运或mRNA在树突内的翻译。该项目产生的对树突蛋白合成的见解将有助于发展

DESCRIPTION (provided by applicant): The brain processes and stores information by constantly adjusting the strength of connections between its neurons. In order to adjust the strength of these connections, called synapses, the neurons have to synthesize new proteins. Proteins can be synthesized in the soma of the neuron and then travel through dendrites to reach the synapses. Alternatively, proteins can be synthesized within the dendrites close to the synapses. This dendritic protein synthesis provides a rapid and efficient way of modifying the strength of single synaptic connections. Dendritic protein synthesis is important for normal brain development and cognitive functions. When dendritic protein synthesis is impaired, for example in Fragile X Syndrome, it causes severe neurodevelopmental and cognitive deficits. Despite its importance, there is an incomplete understanding of dendritic protein synthesis. A major remaining question is: which proteins can be synthesized in dendrites? This project will test and apply novel tools that can answer this question. The tools allow for the selective isolation of ribosome-bound messenger RNA (mRNA) from in-vivo dendrites. Since ribosomes bind to mRNA in order to synthesize proteins through a process called mRNA translation, the ribosome-bound translated mRNA directly reflects which proteins are being synthesized. In order to completely characterize dendritic translated mRNA, this project will use next-generation sequencing in order to sequence each mRNA molecule within the sample (RNA-Seq). Knowing the sequence of each dendritic translated mRNA molecule not only answers the question which proteins can be synthesized in dendrites, but also provides insights into the specific protein isoforms that are synthesized. The sequence data generated by this project can also be used to discover RNA motifs that are shared by groups of dendritic mRNA. Some of these motifs could regulate the transport of mRNA into the dendrite or the translation of mRNA within the dendrite. The insights into dendritic protein synthesis generated by this project will aid the development of treatments for brain disorders associated with impaired dendritic protein synthesis.