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mRNA Processing pp 103–127Cite as

Methods for the Detection of Adenosine-to-Inosine Editing Events in Cellular RNA

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1648))

Abstract

Modification of RNA is essential for properly expressing the repertoire of RNA transcripts necessary for both cell type and developmental specific functions. RNA modifications serve to dynamically re-wire and fine-tune the genetic information carried by an invariable genome. One important type of RNA modification is RNA editing and the most common and well-studied type of RNA editing is the hydrolytic deamination of adenosine to inosine. Inosine is a biological mimic of guanosine; therefore, when RNA is reverse transcribed, inosine is recognized as guanosine by the reverse transcriptase and a cytidine is incorporated into the complementary DNA (cDNA) strand. During PCR amplification, guanosines pair with the newly incorporated cytidines. As a result, the adenosine-to-inosine (A-to-I) editing events are recognized as adenosine to guanosine changes when comparing the sequences of the genomic DNA to the cDNA. This chapter describes the methods for extracting endogenous RNA for subsequent analyses of A-to-I RNA editing using reverse transcriptase-based approaches. We discuss techniques for the detection of A-to-I RNA editing events in messenger RNA (mRNA), including analyzing editing levels at specific adenosines within the total pool of mRNA versus analyzing editing patterns that occur in individual transcripts and a method for detecting editing events across the entire transcriptome. The detection of RNA editing events and editing levels can be used to better understand normal biological processes and disease states.

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References

  1. Gott JM, Emeson RB (2000) Functions and mechanisms of RNA editing. Annu Rev Genet 34:499–531. doi:10.1146/annurev.genet.34.1.499

    Article  CAS  PubMed  Google Scholar 

  2. Licht K, Jantsch MF (2016) Rapid and dynamic transcriptome regulation by RNA editing and RNA modifications. J Cell Biol 213(1):15–22. doi:10.1083/jcb.201511041

  3. Mannion N, Arieti F, Gallo A, Keegan LP, O'Connell MA (2015) New insights into the biological role of mammalian ADARs; the RNA editing proteins. Biomol Ther 5(4):2338–2362. doi:10.3390/biom5042338

    CAS  Google Scholar 

  4. Savva YA, Rieder LE, Reenan RA (2012) The ADAR protein family. Genome Biol 13(12):252. doi:10.1186/gb-2012-13-12-252

    Article  PubMed  PubMed Central  Google Scholar 

  5. Samuel CE (2012) ADARs: viruses and innate immunity. Curr Top Microbiol Immunol 353:163–195. doi:10.1007/82_2011_148

    CAS  PubMed  Google Scholar 

  6. Gommans WM (2012) A-to-I editing of microRNAs: regulating the regulators? Semin Cell Dev Biol 23(3):251–257. doi:10.1016/j.semcdb.2011.09.018

    Article  CAS  PubMed  Google Scholar 

  7. Daniel C, Lagergren J, Ohman M (2015) RNA editing of non-coding RNA and its role in gene regulation. Biochimie 117:22–27. doi:10.1016/j.biochi.2015.05.020

    Article  CAS  PubMed  Google Scholar 

  8. Nishikura K (2016) A-to-I editing of coding and non-coding RNAs by ADARs. Nat Rev Mol Cell Biol 17(2):83–96. doi:10.1038/nrm.2015.4

    Article  CAS  PubMed  Google Scholar 

  9. Li JB, Levanon EY, Yoon JK, Aach J, Xie B, Leproust E, Zhang K, Gao Y, Church GM (2009) Genome-wide identification of human RNA editing sites by parallel DNA capturing and sequencing. Science 324(5931):1210–1213. doi:10.1126/science.1170995

    Article  CAS  PubMed  Google Scholar 

  10. Peng Z, Cheng Y, Tan BC, Kang L, Tian Z, Zhu Y, Zhang W, Liang Y, Hu X, Tan X, Guo J, Dong Z, Liang Y, Bao L, Wang J (2012) Comprehensive analysis of RNA-Seq data reveals extensive RNA editing in a human transcriptome. Nat Biotechnol 30(3):253–260. doi:10.1038/nbt.2122

    Article  CAS  PubMed  Google Scholar 

  11. Porath HT, Carmi S, Levanon EY (2014) A genome-wide map of hyper-edited RNA reveals numerous new sites. Nat Commun 5:4726. doi:10.1038/ncomms5726

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Ramaswami G, Lin W, Piskol R, Tan MH, Davis C, Li JB (2012) Accurate identification of human Alu and non-Alu RNA editing sites. Nat Methods 9(6):579–581. doi:10.1038/nmeth.1982

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Ramaswami G, Zhang R, Piskol R, Keegan LP, Deng P, O'Connell MA, Li JB (2013) Identifying RNA editing sites using RNA sequencing data alone. Nat Methods 10(2):128–132. doi:10.1038/nmeth.2330

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Slotkin W, Nishikura K (2013) Adenosine-to-inosine RNA editing and human disease. Genome Med 5(11):105. doi:10.1186/gm508

    Article  PubMed  PubMed Central  Google Scholar 

  15. Tariq A, Jantsch MF (2012) Transcript diversification in the nervous system: a to I RNA editing in CNS function and disease development. Front Neurosci 6:99. doi:10.3389/fnins.2012.00099

    Article  PubMed  PubMed Central  Google Scholar 

  16. Enstero M, Daniel C, Wahlstedt H, Major F, Ohman M (2009) Recognition and coupling of A-to-I edited sites are determined by the tertiary structure of the RNA. Nucleic Acids Res 37(20):6916–6926. doi:10.1093/nar/gkp731

    Article  PubMed  PubMed Central  Google Scholar 

  17. Wheeler EC, Washburn MC, Major F, Rusch DB, Hundley HA (2015) Noncoding regions of C. elegans mRNA undergo selective adenosine to inosine deamination and contain a small number of editing sites per transcript. RNA Biol 12(2):162–174. doi:10.1080/15476286.2015.1017220

    Article  PubMed  PubMed Central  Google Scholar 

  18. Veno MT, Bramsen JB, Bendixen C, Panitz F, Holm IE, Ohman M, Kjems J (2012) Spatio-temporal regulation of ADAR editing during development in porcine neural tissues. RNA Biol 9(8):1054–1065. doi:10.4161/rna.21082

    Article  PubMed  PubMed Central  Google Scholar 

  19. Bass B, Hundley H, Li JB, Peng Z, Pickrell J, Xiao XG, Yang L (2012) The difficult calls in RNA editing. Interviewed by H Craig Mak. Nat Biotechnol 30(12):1207–1209. doi:10.1038/nbt.2452

    Article  CAS  PubMed  Google Scholar 

  20. Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31(13):3406–3415

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Bazak L, Haviv A, Barak M, Jacob-Hirsch J, Deng P, Zhang R, Isaacs FJ, Rechavi G, Li JB, Eisenberg E, Levanon EY (2014) A-to-I RNA editing occurs at over a hundred million genomic sites, located in a majority of human genes. Genome Res 24(3):365–376. doi:10.1101/gr.164749.113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Alon S, Garrett SC, Levanon EY, Olson S, Graveley BR, Rosenthal JJ, Eisenberg E (2015) The majority of transcripts in the squid nervous system are extensively recoded by A-to-I RNA editing. eLife 4. doi:10.7554/eLife.05198

  23. Fumagalli D, Gacquer D, Rothe F, Lefort A, Libert F, Brown D, Kheddoumi N, Shlien A, Konopka T, Salgado R, Larsimont D, Polyak K, Willard-Gallo K, Desmedt C, Piccart M, Abramowicz M, Campbell PJ, Sotiriou C, Detours V (2015) Principles governing A-to-I RNA editing in the breast cancer transcriptome. Cell Rep 13(2):277–289. doi:10.1016/j.celrep.2015.09.032

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Paz-Yaacov N, Bazak L, Buchumenski I, Porath HT, Danan-Gotthold M, Knisbacher BA, Eisenberg E, Levanon EY (2015) Elevated RNA editing activity is a major contributor to transcriptomic diversity in tumors. Cell Rep 13(2):267–276. doi:10.1016/j.celrep.2015.08.080

    Article  CAS  PubMed  Google Scholar 

  25. Han L, Diao L, Yu S, Xu X, Li J, Zhang R, Yang Y, Werner HM, Eterovic AK, Yuan Y, Li J, Nair N, Minelli R, Tsang YH, Cheung LW, Jeong KJ, Roszik J, Ju Z, Woodman SE, Lu Y, Scott KL, Li JB, Mills GB, Liang H (2015) The genomic landscape and clinical relevance of A-to-I RNA editing in human cancers. Cancer Cell 28(4):515–528. doi:10.1016/j.ccell.2015.08.013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Qiu S, Li W, Xiong H, Liu D, Bai Y, Wu K, Zhang X, Yang H, Ma K, Hou Y, Li B (2016) Single-cell RNA sequencing reveals dynamic changes in A-to-I RNA editome during early human embryogenesis. BMC Genomics 17(1):766. doi:10.1186/s12864-016-3115-2

    Article  PubMed  PubMed Central  Google Scholar 

  27. Washburn MC, Kakaradov B, Sundararaman B, Wheeler E, Hoon S, Yeo GW, Hundley HA (2014) The dsRBP and inactive editor ADR-1 utilizes dsRNA binding to regulate A-to-I RNA editing across the C. elegans transcriptome. Cell Rep 6(4):599–607. doi:10.1016/j.celrep.2014.01.011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgment

Work in the Hundley lab is supported by Research Scholar Award RSG-15-051-01-RMC from the American Cancer Society. We thank Aidan Manning and Dr. Sarah Deffit for helpful comments on the manuscript.

Author information

Authors and Affiliations

  1. Genome, Cell and Developmental Biology Program, Indiana University, Bloomington, IN, USA

    Eimile Oakes

  2. Biotechnology Professional Science Master’s Program, Indiana University, Bloomington, IN, USA

    Pranathi Vadlamani

  3. Medical Sciences Program, Indiana University School of Medicine, Bloomington, IN, USA

    Heather A. Hundley

Authors
  1. Eimile Oakes
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  2. Pranathi Vadlamani
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  3. Heather A. Hundley
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Corresponding author

Correspondence to Heather A. Hundley .

Editor information

Editors and Affiliations

  1. Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, California, USA

    Yongsheng Shi

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Oakes, E., Vadlamani, P., Hundley, H.A. (2017). Methods for the Detection of Adenosine-to-Inosine Editing Events in Cellular RNA. In: Shi, Y. (eds) mRNA Processing. Methods in Molecular Biology, vol 1648. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7204-3_9

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  • DOI: https://doi.org/10.1007/978-1-4939-7204-3_9

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7203-6

  • Online ISBN: 978-1-4939-7204-3

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