DFG - GZ: KI 488/7-1 AOBJ: 629331 Functional analysis of kinesin-associated RNA transport granules in neurons

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.