Functional analysis of kinesin-associated RNA transport granules in neurons

在神经元中，mRNA翻译的动态调节对于神经元细胞生物学的特定方面至关重要，包括突触形成、突触可塑性和从突触到细胞核的信号传导。利用树突定位的mRNA，蛋白质合成可能发生在远离突触附近的索马。为此，特定的mRNA作为大的mRNP转运颗粒转运到树突中。虽然传统的驱动蛋白Kif 5a-c在十多年前就被确定为树突状mRNP的主要载体，但这些颗粒的分子组成、组装和重组以及单个组分在靶mRNA识别、树突状运输和局部翻译控制中的作用仅知之甚少。该项目的目标是Kif 5附着的转运颗粒（KTG）的功能和结构表征。具体而言，我们将（1）使用已建立的方法从啮齿动物脑中纯化KTG，并在转录物和蛋白质水平上提供KTG分子组分的完整表征;（2）通过比较来自核和胞质组分的KTG，分析mRNP形成和成熟的机制。此外，我们将跟踪我们的初步观察，即KTG组成在FMRP缺陷小鼠中改变，从而将KTG功能障碍与神经发育疾病联系起来。在这里，我们将解决的可能性，KTG是由不同的亚复合物，其中一些可能会失去FMRP耗尽。最后（3）我们将确定KTG的各个组分在树突中mRNA转运和定位蛋白质合成方面的分子功能。为此，我们将使用慢病毒shRNA敲低培养的海马神经元中KTG的关键组分，并分析转导细胞的KTG形成、特定树突状mRNA的定位和神经元mRNA的翻译效率。我们项目的长期目标是确定KTG与突触可塑性的功能相关性，并确定KTG功能的破坏如何对突触和可塑性相关行为的功能产生不利影响，从而可能导致神经发育/精神疾病。

In neurons, dynamic regulation of mRNA translation is of critical relevance for specific aspects of neuronal cell biology, including synapse formation, synaptic plasticity and signaling from synapses to the nucleus. Protein synthesis may occur distant from the soma near synapses utilizing dendritically localized mRNAs. For this purpose, specific mRNAs are transported into dendrites as large mRNP transport granules. Although the conventional kinesins Kif5a-c have been identified as major carriers of dendritic mRNPs more than ten years ago, the molecular composition, assembly and restructuring of these granules as well as the role of individual components in target mRNA recognition, dendritic trafficking and local translational control are only sparsely understood. The goal of this project is a functional and structural characterization of Kif5-attached transport granules (KTGs). In particular, we will (1) purify KTGs from rodent brain using established methods, and provide a complete characterization of molecular components of KTGs both on transcript and protein level; (2) analyze mechanisms of mRNP formation and maturation by comparing KTGs derived from nuclear and cytosolic fractions. In addition we will follow up on our initial observation that KTG composition is altered in FMRP deficient mice, thus linking KTG dysfunction to neurodevelopmental disease. Here we will address the possibility that KTGs are made up of different subcomplexes, some of which may be lost upon FMRP depletion. Finally (3) we will determine the molecular function of individual components of KTGs with respect to mRNA transport and localized protein synthesis in dendrites. For this we will knockdown key components of KTGs in cultured hippocampal neurons using lentiviral shRNA, and analyze transduced cells for KTG formation, localization of specific dendritic mRNAs and translation efficiency of neuronal mRNAs. The long-term goal of our project is to identify the functional relevance of KTGs for synaptic plasticity and determine how disruption of KTG function adversely affects the functionality of synapses and plasticity-related behaviours and may thus cause neuro-developmental/-psychiatric disorders.