Mechanistic studies of RNA-binding proteins regulating local RNA translation at synapses

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Neurons are polarized cells that have numerous dendritic processes and an axon that extend away from the cell body. While the majority of neuronal protein synthesis occurs in its somatic compartment, local protein synthesis also occurs in distal regions of the cell. The requirement for regulated protein synthesis at specific locations in neurons is a logistical hurtle that must be overcome to maintain synaptic plasticity and proper neurological functions. To meet the demands of local translation within neurons, mRNAs are bound by RNA binding proteins (RBPs) forming ribonucleoprotein (RNP) complexes that are targeted to specific sites of local translation. We hypothesize that some RBPs have an integral role in regulating local translation in response to intracellular signal transduction pathways. Therefore, the focus of our research program is to determine how RBPs are themselves regulated and how they control local mRNA translation at synapses. While local translation occurs in both pre- and post-synapses of neurons, many questions still remain regarding the regulatory mechanisms of local translation and the role of RBPs in this process. Our program is focused on understanding the mechanisms of how RBPs, like Fused in Sarcoma (FUS), control local translation. While investigating the mammalian target of rapamycin (mTOR) signaling pathway, one of the major signaling pathways that regulates mRNA translation, we observed that pharmacological inhibition of mTOR kinase stimulates the association of FUS with polysomes, the molecular complex that translates mRNA into protein. We have further examined the functional association of FUS with polysomes and found that it associates with stalled polysomes. Consistent with this effect, when we blocked neuronal activity in primary neuron cultures, we observed more co-localization of FUS with ribosomal subunits by super resolution imaging. Taken together, our data suggest that FUS is regulated by intracellular signaling pathways that determine its role in regulating local translation at the synapse. Herein, our program aims to determine how cell signaling pathways regulate FUS to control local translation at synapses. This grant aims to: 1) Determine how cell signaling pathways, which impact translation, regulate FUS activity at the synapse; 2) Determine the post-translational regulation of FUS and the impact on local translation; and 3) Identify the FUS protein-protein and protein-RNA interactions in synaptic polysomes in response to translation inhibition or activation. Findings from this proposal will act as a stepping stone towards the long term goal of our research program, which is to determine how local translation controls synaptic plasticity over the life-time of a neuron. We hope our proposed studies will provide novel insights into the regulatory mechanisms that control local protein expression and lead to the development of new ways to control local translation.