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C-to-U RNA Editing: From Computational Detection to Experimental Validation

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RNA Editing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2181))

Abstract

The AID/APOBEC family of enzymes are cytidine deaminases that act upon DNA and RNA. Among APOBECs, the best characterized family member to act on RNA is the enzyme APOBEC1. APOBEC1-mediated RNA editing plays a key role in lipid metabolism and in maintenance of brain homeostasis. Editing can be easily detected in RNA-seq data as a cytosine to thymine (C-to-T) change with regard to the reference. However, there are many other sources of base conversions relative to reference, such as PCR errors, SNPs, and even DNA editing by mutator APOBECs. Furthermore, APOBEC1 exhibits disparate activity in different cell types, with respect to which transcripts are edited and the level to which they are edited. When considering these potential sources of error and variability, an RNA-seq comparison between wild-type APOBEC1 sample and a matched control with an APOBEC1 knockout is a reliable method for the discrimination of true sites edited by APOBEC1. Here we present a detailed description of a method for studying APOBEC1 RNA editing, specifically in the murine macrophage cell line RAW 264.7. Our method covers the production of an APOBEC1 knockout cell line using the CRISPR/Cas9 system, through to experimental validation and quantification of editing sites (where we discuss a recently published algorithm (termed MultiEditR) which allows for the detection and quantification of RNA editing from Sanger sequencing). Importantly, this same protocol can be adapted to any RNA modification detectable by RNA-seq analysis for which the responsible protein is known.

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Acknowledgments

We thank the Flow Cytometry unit of the Imaging and Cytometry Core Facility, German Cancer Research Center (DKFZ), for providing excellent sorting services. We also thank Derek Nedveck (University of Minnesota) for his help with questions surrounding R shiny. Finally we also thank Walker Lahr (University of Minnesota) for helpful conversations surrounding this topic.

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Author notes

  1. Taga Lerner, Mitchell Kluesner, Rafail Nikolaos Tasakis, Branden S. Moriarity, F. N. Papavasiliou and Riccardo Pecori contributed equally with all other contributors.

Authors and Affiliations

  1. Division of Immune Diversity, Program in Cancer Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany

    Taga Lerner, Rafail Nikolaos Tasakis, F. Nina Papavasiliou & Riccardo Pecori

  2. Faculty of Biosciences, Heidelberg University, Heidelberg, Germany

    Taga Lerner & Rafail Nikolaos Tasakis

  3. Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA

    Mitchell Kluesner & Branden S. Moriarity

  4. Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA

    Mitchell Kluesner & Branden S. Moriarity

  5. Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA

    Mitchell Kluesner & Branden S. Moriarity

Authors
  1. Taga Lerner
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  2. Mitchell Kluesner
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  3. Rafail Nikolaos Tasakis
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  4. Branden S. Moriarity
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  5. F. Nina Papavasiliou
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  6. Riccardo Pecori
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Corresponding author

Correspondence to Riccardo Pecori .

Editor information

Editors and Affiliations

  1. Department of Biosciences, Biotechnology and Biopharmaceutics, Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies, National Research Council, University of Bari “Aldo Moro”, Bari, Italy, Bari, Italy

    Ernesto Picardi

  2. Department of Biosciences, Biotechnology and Biopharmaceutics, Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies, National Research Council, University of Bari “Aldo Moro”, Bari, Italy, Bari, Italy

    Graziano Pesole

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Lerner, T., Kluesner, M., Tasakis, R.N., Moriarity, B.S., Papavasiliou, F.N., Pecori, R. (2021). C-to-U RNA Editing: From Computational Detection to Experimental Validation. In: Picardi, E., Pesole, G. (eds) RNA Editing. Methods in Molecular Biology, vol 2181. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0787-9_4

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  • DOI: https://doi.org/10.1007/978-1-0716-0787-9_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0786-2

  • Online ISBN: 978-1-0716-0787-9

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