Regulation of local translation in glia

摘要 为了实现有效的专门化和亚细胞区域的动态调节，许多细胞在局部进化 信使核糖核酸的翻译--然而星形胶质细胞中这种翻译调控的基本原理尚不清楚。 在神经元中进行了长期的研究，各种蛋白质的局部翻译被认为是突触的关键- 作为学习和记忆基础的特定变化。在本裁决的前一个周期中，我们提供了证据 星形胶质细胞也通过各种途径调节局部翻译。在这里，我们建议继续 关于这一令人着迷的新的基本生物学现象，这项工作着重于以下问题： 是不是调控语法决定了星形胶质细胞何时以及哪些转录产物被局部翻译？ 我们的中心模型是，转录本的非翻译区(UTRs)中的元素负责其 外周星形胶质细胞突起(PAP)中核糖体的浓缩或枯竭，通过与UTR的相互作用 RNA结合蛋白(RBPs)和microRNAs(MiRNAs)。然而，由于有数百个潜在的元素 筛选、新的、可扩展的方法需要系统地表征RNA的本地化和翻译 在星形胶质细胞中被调节，在基线和对信号提示的反应中都是如此。此外，只有神经胶质细胞形态 在体内达到完全成熟，需要进行体内功能研究。因此，我们开发了一种新的方法，一种 突触神经体大规模平行报告分析(SN-MPRA)使我们能够评估功能效应 体内同时存在数千个候选的非编码区元件。我们将应用这一点来定义 调节星形胶质细胞中的RNA定位。此外，为了更好地理解这些元素的子集是如何 我们将定义一个特定的RBP‘quking’(QKI)在调节星形胶质细胞局部翻译中的作用。 最后，为了了解如何以协调一致的方式管理成套成绩单以供当地翻译， 我们将研究miRNA效应蛋白(Ago2)和特定的miRNAs在调节局部 星形胶质细胞中的翻译。

ABSTRACT To enable efficient specialization and dynamic regulation of subcellular regions, many cells have evolved local translation of mRNA - yet the fundamental principles of such translation regulation in astrocytes are unknown. Long studied in neurons, local translation of a variety of proteins is thought to be essential for the synapse- specific changes that underlay learning and memory. In the prior cycle of this award we provided evidence astrocytes also have a regulated local translation by using a variety of approaches. Here, we propose to continue this work, focusing on the following question regarding this fascinating new basic biological phenomenon: what is the regulatory grammar that determines when and which transcripts are locally translated in astrocytes? Our central model is that elements in the untranslated regions (UTRs) of transcripts are responsible for their enrichment or depletion from ribosomes in peripheral astrocyte processes (PAPs), via UTR interactions with RNA binding proteins (RBPs) and microRNAs (miRNAs). However, with hundreds of potential elements to screen, new, scalable methods are needed to systematically characterize how RNA localization and translation is regulated in astrocytes, both at baseline and in response to signaling cues. Furthermore, glial morphology only reaches full maturity in vivo, requiring in vivo functional studies. Therefore, we have developed a new method, a synaptoneurosome–massively parallel reporter assay (SN-MPRA) which allows us to assess functional effects of thousands of candidate UTR elements in vivo simultaneously. We will apply this to define the sequences that modulate RNA localization in astrocytes. Furthermore, to better understand how a subset of these elements function, we will define the role of a specific RBP, ‘quaking’ (QKI), in modulating local translation in astrocytes. Finally, to understand how sets of transcripts might be regulated in a coordinated fashion for local translation, we will examine the role of miRNA effector proteins (Ago2) along with specific miRNAs in regulating local translation in astrocytes.