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

描述(申请人提供)：大脑通过不断调整其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。或者，蛋白质可以在靠近突触的树突中合成。这种树突状蛋白的合成提供了一种快速而有效的方式来改变单个突触连接的强度。树突状蛋白的合成对正常的大脑发育和认知功能非常重要。当树突状蛋白合成受损时，例如在脆性X综合征中，它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，但人们对它的了解还不够全面。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能够回答这个问题的新工具。这些工具允许从体内树突中选择性地分离核糖体结合的信使RNA(MRNA)。由于核糖体与信使核糖核酸结合，以便通过一种称为信使核糖核酸翻译的过程来合成蛋白质，因此，核糖体结合的已翻译信使核糖体直接反映了正在合成的蛋白质。为了完整地表征树突状翻译的mRNA，该项目将使用下一代测序来对样本中的每个mRNA分子进行测序(RNA-Seq)。了解每个树突翻译的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亚型的洞察。该项目产生的序列数据也可以用来发现树突状mRNA组共享的RNA基序。其中一些基序可以调节mRNA在树突中的转运或在树突中的翻译。该项目对树突状蛋白质合成的洞察力将有助于 治疗与树突状蛋白合成受损相关的脑部疾病。

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.