Addressing protein synthesis regulation within small numbers of discrete neurons

项目总结/摘要 最近的出版物表明，转录后调控对基因的表达有重要的贡献。 昼夜节律，在苍蝇和哺乳动物中。这些证据现在相当广泛，包括 翻译调控，影响哺乳动物SCN内关键时钟蛋白的合成， 以及控制果蝇昼夜运动的少数中枢神经元 活动节律这些神经元对昼夜节律生物学提出了重大挑战--也是机遇， 对于分子生物学来说，更普遍的是：如何评估几个神经元内的生化事件？为 几乎所有的测量，没有足够的信号和/或有问题的信噪比时， 制备提取物并从仅几个细胞/脑开始测定。即使是标记大脑神经元的方法， 如果标记的神经元/脑的分数非常低，则会受到这个问题的困扰。为了解决这个问题， 在RNA结合蛋白及其靶点的背景下，我的实验室开发了一种新技术，我们称之为 TRIBE（通过编辑识别的RNA结合蛋白的靶标）。它融合了RNA的催化结构域 将编辑酶阿达尔转化为RNA结合蛋白。部落利用了一个人可以 清洁的RNA和序列，即使从一个单一的细胞，尽管无法与提取物的工作， 相同的源材料。我们已经使用了TRIBE与三种不同的RBP，也表明它可以工作， 在“少量特定神经元”的背景下。我们最近验证了TRIBE并改进了其 功效，因此它具有更少的假阴性，即，它识别的RBP比例要大得多 相关的mRNA。我们将在两个新的以预防为中心的方向上扩展该方法： 重要翻译因子eIF 4 E-BP的特异性靶点，并鉴定核糖体相关的 成绩单我们还建议解决一些剩余的问题，并将TRIBE扩展到新的方向， CRISPR介导的敲入以及二聚化方法。后者将带来编辑 RNA的分子量，即RNA分子量。响应于药物或光介导的二聚化信号。在所有 在许多情况下，成功地分析少量离散神经元是关键的生物焦点。这是一 与许多人类健康问题如精神疾病、神经变性、中风 药物滥用和成瘾。

Project Summary/Abstract Recent publications indicate that post-transcriptional regulation makes an important contribution to circadian rhythms, in flies and in mammals. This evidence is now quite extensive and includes translational regulation, which impacts the synthesis of key clock proteins within the mammalian SCN as well as within the small number of central brain neurons that govern Drosophila circadian locomotor activity rhythms. These neurons pose a significant challenge – and opportunity – for circadian biology and for molecular biology more generally: how can one assess biochemical events within a few neurons? For almost all measurements, there is not enough signal and/or a problematic signal:noise ratio when an extract is made and assayed starting with only a few cells/brain. Even methods that tag brain neurons are plagued with this problem if the fraction of tagged neurons/brain is very low. To address this issue in the context of RNA binding proteins and their targets, my lab developed a new technique, which we call TRIBE (Targets of RNA Binding proteins Identified by Editing). It fuses the catalytic domain of the RNA editing enzyme ADAR to RNA binding proteins. TRIBE takes advantage of the fact that one can make clean RNA and sequence it even from a single cell despite the inability to work with an extract from the same source material. We have used TRIBE with three different RBPs and also showed that it can work within the “small number of specific neurons” context. We have recently validated TRIBE and improved its efficacy, so that it has many fewer false negatives, i.e., it recognizes a much greater fraction of RBP- associated mRNAs. We will extend the method in two new translation-centric directions: to identify specific targets of the important translation factor eIF4E-BP and to identify ribosome-associated transcripts. We also propose to address some remaining issues, and extend TRIBE in new directions, with CRISPR-mediated knock-ins as well as with dimerization approaches. The latter will bring the editing moiety to the RNA on command, i.e., in response to a drug- or light-mediated dimerization signal. In all cases, assaying successfully small numbers of discrete neurons is the key biological focus. It is an important goal relevant to many human health problems like mental illness, neurodegeneration, stroke, substance abuse and addiction.