Subcellular localization of mRNAs plays an important role for regulation of local protein synthesis, especially in the context of development and neuronal plasticity. One class of RNA binding proteins are DBHS proteins, consisting of three homologous proteins NONO, PSPC1 and SFPQ, each of which contains two RNA recognition motifs (RRMs). DBHS proteins have been implicated in a wide range of functions, such as transcriptional activation and repression, RNA splicing, and regulation of RNA nuclear export. Moreover, they have been identified as components of both nuclear paraspeckles and synaptic RNA transport granules. However, the role of RNA binding proteins in synaptic morphology and plasticity is only poorly understood. This project examines the role of RNA binding proteins of the DBHS family in regulating inhibitory synapse function, and in particular its function within the circadian clock.
Figure 1: GFP-NONO (green) localizes to paraspeckles in the nucleus (blue) of primary rat hippocampal neurons.
To elucidate the role of these proteins in regulation of synaptic signaling, we investigated whether they influence the assembly and stability of the postsynaptic density. An important component of the macromolecular complex at inhibitory synapses is Gephyrin, a highly conserved multifunctional scaffolding protein. Gain of function experiments strongly suggest that NONO and PSPC1 can affect gephyrin clustering. To continue with these experiments, RNAi constructs for each DBHS protein will be transfected into primary neurons as above in order to determine effects of loss of function of each of these three proteins upon gephyrin polymerization and synaptic concentration of GABA receptor subtypes. Finally, the relevance of these effects will be tested in vivo by looking at gephyrin structure and GABA receptor distribution at hippocampal synapses in fixed brain slices from mice deleted for each of the DBHS protein.
Figure 2: Cultured primary rat hippocampal neurons transfected with GFP-Gephyrin (green) colocalize with endogenious Gepyrin (red) at postsynaptic inhibitory sites.