Abstract
Paired-end RNA sequencing (RNA-seq) is usually applied to the quantification of long transcripts such as messenger or long non-coding RNAs, in which case overlapping pairs are discarded. In contrast, RNA-seq on short RNAs (≤ 200 nt) is typically carried out in single-end mode, as the additional cost associated with paired-end would only translate into redundant sequence information. Here, we exploit paired-end sequencing of short RNAs as a strategy to filter out sequencing errors and apply this method to the identification of adenosine-to-inosine (A-to-I) RNA editing events on human precursor microRNA (pre-miRNA) and mature miRNA. Combined with RNA immunoprecipitation sequencing (RIP-seq) of A-to-I RNA editing enzymes, this method takes full advantage of deep sequencing technology to identify RNA editing sites with unprecedented resolution in terms of editing efficiency.
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References
Ihle MA, Fassunke J, König K, Grünewald I, Schlaak M, Kreuzberg N, Tietze L, Schildhaus HU, Büttner R, Merkelbach-Bruse S (2014) Comparison of high resolution melting analysis, pyrosequencing, next generation sequencing and immunohistochemistry to conventional sanger sequencing for the detection of p.V600E and non-p.V600E BRAF mutations. BMC Cancer 14:13
Kawahara Y, Zinshteyn B, Sethupathy P, Iizasa H, Hatzigeorgiou AG, Nishikura K (2007) Redirection of silencing targets by adenosine-to-inosine editing of miRNAs. Science 315(5815):1137–1140
Kume H, Hino K, Galipon J, Ui-Tei K (2014) A-to-I editing in the miRNA seed region regulates target mRNA selection and silencing efficiency. Nucleic Acids Res 42:10050–10060
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 massively parallel DNA capturing and sequencing. Science 324:1210–1213 *equal contribution
Bahn JH, Lee JH, Li G, Greer C, Peng G, Xiao X (2012) Accurate identification of A-to-I RNA editing in human by transcriptome sequencing. Genome Res 22:142–150
Chepelev I (2012) Detection of RNA editing events in human cells using high-throughput sequencing. Methods Mol Biol 815:91–102
Galipon J, Ishii R, Suzuki Y, Tomita M, Ui-Tei K (2017) Differential binding of three major human ADAR isoforms to coding and long non-coding transcripts. Genes (Basel) 8(2):68
Hafner M, Landthaler M, Burger L, Khorshid M, Hausser J, Berninger P, Rothballer A, Ascano M, Jungkamp AC, Munschauer M, Ulrich A, Wardle GS, Scott Dewell S, Zavolan Z, Tuschl T (2010) J Vis Exp 41:2034–2039
Chi SW, Zang JB, Mele A, Darnell RB (2009) Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460:479–486
Weeks KM, Crothers DM (1993) Major groove accessibility of RNA. Science 261:1574–1577
Liu ZR, Sargueil B, Smith CW (2000) Methylene blue-mediated cross-linking of proteins to double-stranded RNA. Methods Enzymol 318:22–33
Tuite EM, Kelly JM (1993) Photochemical interactions of methylene blue and analogues with DNA and other biological substrates. J Photochem Photobiol B 21:103–124
Wu D, Lamm AT, Fire AZ (2011) Competition between ADAR and RNAi pathways for an extensive class of RNA targets. Nat Struct Mol Biol 18:1094–1101
1000 Genomes Project Consortium, Abecasis GR, Auton A, Brooks LD, MA DP, Durbin RM, Handsaker RE, Kang HM, Marth GT, McVean GA (2012) An integrated map of genetic variation from 1,092 human genomes. Nature 491:56–65
Sherry ST, Ward MH, Kholodov M, Baker J, Phan L, Smigielski EM, Sirotkin K (2001) dbSNP: the NCBI database of genetic variation. Nucleic Acids Res 29:308–311
The International HapMap Consortium (2003) The international HapMap project. Nature 426:789–796
Gong J, Liu C, Liu W, Wu Y, Ma Z, Chen H, Guo AY 2015 An update of miRNASNP database for better SNP selection by GWAS data, miRNA expression and online tools. Database (Oxford) 2015:bav029
Han M, Zheng Y (2013) Comprehensive analysis of single nucleotide polymorphisms in human microRNAs. PLoS One 8:e78028
Kawahara Y, Megraw M, Kreider E, Iizasa H, Valente L, Hatzigeorgiou AG, Nishikura K (2008) Frequency and fate of microRNA editing in human brain. Nucleic Acids Res 36:5270–5280
Luciano DJ, Mirsky H, Vendetti NJ, Maas S (2004) RNA editing of a miRNA precursor. RNA 10:1174–1177
García-López J, Hourcade Jde D, Del Mazo J (2013) Reprogramming of microRNAs by adenosine-to-inosine editing and the selective elimination of edited microRNA precursors in mouse oocytes and preimplantation embryos. Nucleic Acids Res 41:5483–5493
Ekdahl Y, Farahani HS, Behm M, Lagergren J, Öhman M (2012) A-to-I editing of microRNAs in the mammalian brain increases during development. Genome Res 22:1477–1487
Alon S, Mor E, Vigneault F, Church GM, Locatelli F, Galeano F, Gallo A, Shomron N, Eisenberg E (2012) Systematic identification of edited microRNAs in the human brain. Genome Res 22:1533–1540
Ramaswami G, Li JB (2014) RADAR: a rigorously annotated database of A-to-I RNA editing. Nucleic Acids Res 42(Database issue):D109–D113
Warnefors M, Liechti A, Halbert J, Valloton D, Kaessmann H (2014) Conserved microRNA editing in mammalian evolution, development and disease. Genome Biol 15:R83
Wang Y, Xu X, Yu S, Jeong KJ, Zhou Z, Han L, Tsang YH, Li J, Chen H, Mangala LS, Yuan Y, Eterovic AK, Lu Y, Sood AK, Scott KL, Mills GB, Liang H (2017) Systematic characterization of A-to-I RNA editing hotspots in microRNAs across human cancers. Genome Res 27(7):1112-1125
Tomaselli S, Galeano F, Alon S, Raho S, Galardi S, Polito V, Presutti C, Vincenti S, Eisenberg E, Locatelli F, Gallo A (2015) Modulation of microRNA editing, expression and processing by ADAR2 deaminase in glioblastoma. Genome Biol 16(1):5
Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25
Bu D, Yu K, Sun S, Xie C, Skogerbø G, Miao R, Xiao H, Liao Q, Luo H, Zhao G, Zhao H, Liu Z, Liu C, Chen R, Zhao Y (2012) NONCODE v3.0: integrative annotation of long noncoding RNAs. Nucleic Acids Res 40:D210–D215
Kozomara A, Griffiths-Jones S (2011) miRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res 39:D152–D157
Neilson JR, Sandberg R (2010) Heterogeneity in mammalian RNA 3′ end formation. Exp Cell Res 316:1357–1364
Galipon J, Ishiguro S, Tomita M, Ui-Tei K (2016) Detection of A-to-I RNA editing by RNA-Seq. NGS application, Handbook of RNA-Seq experiment, 113–117, Yodosha.
Acknowledgments
This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan to KU-T. Keio University Institute for Advanced Bioscience affiliates were supported by research funds from the Yamagata prefectural government and the City of Tsuruoka.
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Galipon, J. et al. (2018). High-Quality Overlapping Paired-End Reads for the Detection of A-to-I Editing on Small RNA. In: Ørom, U. (eds) miRNA Biogenesis. Methods in Molecular Biology, vol 1823. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-8624-8_13
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DOI: https://doi.org/10.1007/978-1-4939-8624-8_13
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