Circular RNAs: novel regulators of dendritic protein synthesis during mammalian synapse development

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The precise regulation of local protein synthesis in neuronal dendrites is essential for activity-dependent synapse formation, remodeling and plasticity, and defects in this process are linked to neurodevelopmental disorders, including mental retardation and autism. Research during the last decade has identified microRNAs (miRNAs), an extensive class of non-coding RNAs, and RNA-binding proteins (RBP) as important regulators of local protein synthesis and dendrite morphogenesis in neurons. However, how miRNA/RBP function itself is orchestrated by neuronal activity is largely uknown. During the first funding period, we focused the role of a miRNA/RBP interplay in the regulation of dendrite morphology. Thereby, we identified two RBPs, Nova1 and Ncoa3, that are required for the repressive activity of dendritic miRNAs. Whereas Nova1 is part of neuronal miRISC, Ncoa3 indirectly stimulates miRISC function by inducing Ago2 expression (Störchel et al., EMBO J. 2015). We further identified a competing endogenous RNA, Ube3a-1, as an additional non-coding regulatory mechanism that controls the availability of specific dendritic miRNAs (Valluy et al., Nat. Neurosci 2015). In the second funding period, we plan to focus on circular RNAs (circRNAs), a subclass of long non-coding RNAs that are generated by specific head-to-tail splicing events (backsplicing). In preliminary experiments, we found that circRNAs in rat hippocampal neurons were abundant, highly enriched in the dendritic compartment and regulated by neuronal activity. RNAi-mediated knockdown of specific circRNAs impaired BDNF-dependent dendritogenesis, suggesting their involvement in neural circuit development. In silico analysis indicated the presence of translational start sites and RBP binding motifs in specific dendritic circRNAs, providing a rationale for further mechanistic studies. Together, this led us to hypothesize that circRNAs are important regulators of dendritic protein synthesis and activity-dependent synapse development. To address this hypothesis, we will pursue the following specific aims: (1) to comprehensively validate dendritic localization of candidate circRNAs identified by RNAseq in vitro and in vivo; (2) to interrogate the function of up to 24 candidate circRNAs in activity-dependent synapse development and plasticity of hippocampal neurons using RNAi; (3) to unravel the internal sequence and structure of dendritic circRNAs by long RNA-seq approaches; (4) to identify the mechanism underlying dendritic circRNA function, with a focus on miRNA/RBP sponges, dendrite delivery vehicles and mRNA traps. By addressing these aims, we will obtain unprecedented insight into the role of circRNAs in mammalian synapse development and plasticity. This will provide a framework for future studies that will address circRNA function in a more physiological context in the intact brain.