Screening and Characterization of Functional circRNAs in Neuronal Cultures

Abstract

This chapter describes a methodology for the screening and characterization of functional circRNAs, particularly in the context of neural circuit development. Taking advantage of a primary rat neuron culture model of synaptogenesis, we propose a means of selecting from the plethora of circRNA species based on their expression levels, dendritic localization, conservation, and activity regulation. These candidates are then knocked down with RNAi approaches in a functional screen for their potential role in the formation and maturation of excitatory synapses.Upon identification of top candidates regulating synaptogenesis, we tie together different "Omics" approaches to explore the molecular mechanisms underlying the phenotypes observed upon circRNA knockdown. We utilized our EnrichMir algorithm to identify overrepresented miRNA binding sites in differentially expressed genes from polyA-RNA-seq following circRNA knockdown. Furthermore, our ScanMiR web tool allows for the miRNA binding prediction of reconstructed internal circular RNA sequences. Small-RNA sequencing is used to monitor changes in miRNA levels in the circRNA knockdown to complement results obtained from EnrichMiR. Finally, the experimental validation of promising miRNA-circRNA pairs sets the stage for in-depth biochemical exploration of the circRNA interactome and mechanism of action.

Keywords: Activity regulation; Binding site discovery; Circular RNA; Compartment-enriched circRNAs; Dendritic processes; EnrichMir; Enrichment; Functional screen; Molecular mechanism; Neurons; PolyA-seq; RNA interference; ScanMir; Small-RNA-seq; Synapses; microRNA.