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Screening and Characterization of Functional circRNAs in Neuronal Cultures

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Circular RNAs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2765))

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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.

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Acknowledgments

We would like to thank Christoph Dieterich and Tobias Jacobi for their expertise in circRNA reconstruction from RNA-sequencing datasets and for the statistical assessment of differentially expressed circRNAs in neuronal compartments. We thank Michael Soutschek, Tomás Germade, and Pierre-Luc Germain for their essential development and maintenance of the ScanMir and EnrichMir tools. We would also like to thank David Colameo for the development of automated synapse morphology analysis pipelines and Silvia Bicker for the optimization of circRNA targeting by RNAi. Figures were created with BioRender.com.

This work was funded by a PhD fellowship to D.K. from the Swiss National Science Foundation (SNSF), NCCR “RNA and Disease.”

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Authors and Affiliations

  1. Lab of Systems Neuroscience, Institute for Neuroscience, Department of Health Science and Technology, Swiss Federal Institute of Technology ETH, Zurich, Switzerland

    Darren Kelly & Gerhard Schratt

Authors
  1. Darren Kelly
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  2. Gerhard Schratt
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Correspondence to Gerhard Schratt .

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Editors and Affiliations

  1. Klaus Tschira Institute for Integrative Computational Cardiology, University Heidelberg, Heidelberg, Germany

    Christoph Dieterich

  2. Department of Cellular, Computational and Integrative Biology – CIBIO, University of Trento, Trento, Italy

    Marie-Laure Baudet

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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Kelly, D., Schratt, G. (2024). Screening and Characterization of Functional circRNAs in Neuronal Cultures. In: Dieterich, C., Baudet, ML. (eds) Circular RNAs. Methods in Molecular Biology, vol 2765. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3678-7_17

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  • DOI: https://doi.org/10.1007/978-1-0716-3678-7_17

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3677-0

  • Online ISBN: 978-1-0716-3678-7

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