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High-Throughput Small RNA Sequencing Enhanced by AlkB-Facilitated RNA de-Methylation (ARM-Seq)

  • Eva Hrabeta-Robinson
  • Erin Marcus
  • Aaron E. Cozen
  • Eric M. Phizicky
  • Todd M. Lowe
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1562)

Abstract

N1-methyladenosine (m1A), N3-methylcytidine (m3C), and N1-methylguanosine (m1G) are common in transfer RNA (tRNA) and tRNA-derived fragments. These modifications alter Watson-Crick base-pairing, and cause pauses or stops during reverse transcription required for most high-throughput RNA sequencing protocols, resulting in inefficient detection of methyl-modified RNAs. Here, we describe a procedure to demethylate RNAs containing m1A, m3C, or m1G using the Escherichia coli dealkylating enzyme AlkB, along with instructions for subsequent processing with widely used protocols for small RNA sequencing.

Key words

RNA Sequencing Transfer RNA (tRNA) AlkB RNA demethylation N1-methyladenosine (m1A) N3-methylcytidine (m3C) N1-methylguanosine (m1G) 

Notes

Acknowledgments

This work was supported by the US National Institutes of Health (NIH) NHGRI grant 5R01HG006753 to T.M.L. and by NIH grant GM052347 to E.M.P. Thanks are given to Patricia Chan for the assistance in the preparation of the figures.

References

  1. 1.
    Machnicka MA, Milanowska K, Osman Oglou O, Purta E, Kurkowska M, Olchowik A, Januszewski W, Kalinowski S, Dunin-Horkawicz S, Rother KM, Helm M, Bujnicki JM, Grosjean H (2013) MODOMICS: a database of RNA modification pathways--2013 update. Nucleic Acids Res 41(Database issue):D262–D267. doi: 10.1093/nar/gks1007 CrossRefPubMedGoogle Scholar
  2. 2.
    Motorin Y, Muller S, Behm-Ansmant I, Branlant C (2007) Identification of modified residues in RNAs by reverse transcription-based methods. Methods Enzymol 425:21–53. doi: 10.1016/S0076-6879(07)25002-5 CrossRefPubMedGoogle Scholar
  3. 3.
    Delaney JC, Essigmann JM (2004) Mutagenesis, genotoxicity, and repair of 1-methyladenine, 3-alkylcytosines, 1-methylguanine, and 3-methylthymine in alkB Escherichia coli. Proc Natl Acad Sci U S A 101(39):14051–14056. doi: 10.1073/pnas.0403489101 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Aas PA, Otterlei M, Falnes PO, Vagbo CB, Skorpen F, Akbari M, Sundheim O, Bjoras M, Slupphaug G, Seeberg E, Krokan HE (2003) Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA. Nature 421(6925):859–863. doi: 10.1038/nature01363 CrossRefPubMedGoogle Scholar
  5. 5.
    Falnes PO (2004) Repair of 3-methylthymine and 1-methylguanine lesions by bacterial and human AlkB proteins. Nucleic Acids Res 32(21):6260–6267. doi: 10.1093/nar/gkh964 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    D’Silva S, Haider SJ, Phizicky EM (2011) A domain of the actin binding protein Abp140 is the yeast methyltransferase responsible for 3-methylcytidine modification in the tRNA anti-codon loop. RNA 17(6):1100–1110. doi: 10.1261/rna.2652611 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Cozen AE, Quartley E, Holmes AD, Hrabeta-Robinson E, Phizicky EM, Lowe TM (2015) ARM-seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments. Nat Methods 12(9):879–884. doi: 10.1038/nmeth.3508 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15(12):550. doi: 10.1186/s13059-014-0550-8 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Quartley E, Alexandrov A, Mikucki M, Buckner FS, Hol WG, DeTitta GT, Phizicky EM, Grayhack EJ (2009) Heterologous expression of L. major proteins in S. cerevisiae: a test of solubility, purity, and gene recoding. J Struct Funct Genomics 10(3):233–247. doi: 10.1007/s10969-009-9068-9 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Eva Hrabeta-Robinson
    • 1
  • Erin Marcus
    • 2
  • Aaron E. Cozen
    • 1
  • Eric M. Phizicky
    • 2
  • Todd M. Lowe
    • 3
  1. 1.Department of Biomolecular EngineeringUniversity of California Santa CruzSanta CruzUSA
  2. 2.Department of Biochemistry & Biophysics, Center for RNA BiologyUniversity of Rochester School of MedicineRochesterUSA
  3. 3.Department of Biomolecular EngineeringUniversity of California Santa CruzSanta CruzUSA

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