Synaptogenesis and synaptic plasticity rely on protein synthesis, in particular on localized translation within dendrites. The aim of my project will focus on investigating the multiple and overlapping molecular mechanisms that contribute to fine-tuning the expression of dendritic mRNA at synapses. In my previous work, I have identified a novel regulatory pathway that involves splicing and modulates the stability and expression of a subset of neuronal and dendritic mRNAs. Prominent amongst the mRNAs analyzed in my studies is Arc, encoding a protein essential for memory consolidation and maintenance of LTP. Several approaches will be undertaken to elucidate the molecular elements controlling Arc mRNA expression: 1) In vivo biochemical isolation and characterization of the ribonucleoprotein particle associated with Arc mRNA 3’UTR region. The procedure will be designed in order to identify trans-acting factors whose binding is connected to: i) synaptic activity ii) dendritic transport iii) splicing of the mRNA. 2) To unveil the role of the identified factors, assays will be carried out in vivo and in cultured neurons, and will include: silencing via lentiviral expression of siRNAs, microscopy, electrophysiology and expression of reporter Arc genes carrying specific modifications. 3) Involvement of miRNAs and of cis-acting elements in controlling synaptic Arc protein expression will also be tested. miRNAs-dependent regulation will be verified in cultured neurons and potential coupling with other pathways of translational and mRNA stability regulation will be tested. Overall, the in vivo biochemical and functional characterization of both cis- and trans-acting factors controlling the expression of Arc mRNA, will allow a better understanding of the mechanisms underlying its modulation, and likely shed light on the pathways controlling dendritic mRNA localized expression and synaptic plasticity.
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