Illustrating the Epitranscriptome at Nucleotide Resolution Using Methylation-iCLIP (miCLIP)

  • Harry George
  • Jernej Ule
  • Shobbir HussainEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1562)


Next-generation sequencing technologies have enabled the transcriptome to be profiled at a previously unprecedented speed and depth. This yielded insights into fundamental transcriptomic processes such as gene transcription, RNA processing, and mRNA splicing. Immunoprecipitation-based transcriptomic methods such as individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) have also allowed high-resolution analysis of the RNA interactions of a protein of interest, thus revealing new regulatory mechanisms. We and others have recently modified this method to profile RNA methylation, and we refer to this customized technique as methylation-iCLIP (miCLIP). Variants of miCLIP have been used to map the methyl-5-cytosine (m5C) or methyl-6-adenosine (m6A) modification at nucleotide resolution in the human transcriptome. Here we describe the m5C-miCLIP protocol, discuss how it yields the nucleotide-resolution RNA modification maps, and comment on how these have contributed to the new field of molecular genetics research coined “epitranscriptomics.”

Key words

Epitranscriptome Epitranscriptomics RNA methylation Methylation-iCLIP miCLIP NSun2 



We wish to acknowledge Dr. Julian Konig who codeveloped the original iCLIP protocol, and Dr. Yoichiro Sugimoto for helpful feedback and discussions during the development of methylation-iCLIP. Research in the SH laboratory is supported by a Seed Award in Science from the Wellcome Trust (WT108285MA), and a Responsive Mode Project Grant from the Biotechnology and Biosciences Research Council (BBSRC) UK (BB/N000749/1).


  1. 1.
    Dubin DT, Taylor RH (1975) The methylation state of poly A-containing messenger RNA from cultured hamster cells. Nucleic Acids Res 2:1653–1668CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S et al (2012) Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485:201–206CrossRefPubMedGoogle Scholar
  3. 3.
    Meyer KD, Saletore Y, Zumbo P, Elemento O, Mason CE, Jaffrey SR (2012) Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons. Cell 149:1635–1646CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Squires JE, Patel HR, Nousch M, Sibbritt T, Humphreys DT, Parker BJ et al (2012) Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA. Nucleic Acids Res 40:5023–5033CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Khoddami V, Cairns BR (2013) Identification of direct targets and modified bases of RNA cytosine methyltransferases. Nat Biotechnol 31:458–464CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Hussain S, Sajini AA, Blanco S, Dietmann S, Lombard P, Sugimot Y et al (2013a) NSun2-mediated cytosine-5 methylation of vault noncoding RNA determines its processing into regulatory small RNAs. Cell Rep 4:255–261CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Wang X, Lu Z, Gomez A, Hon GC, Yue Y, Han D et al (2014) N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505:117–120CrossRefPubMedGoogle Scholar
  8. 8.
    Wang X, Zhao BS, Roundtree IA, Lu Z, Han D, Ma H et al (2015) N(6)-methyladenosine modulates messenger RNA translation efficiency. Cell 161:1388–1399CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Meyer KD, Patil DP, Zhou J, Zinoviev A, Skabkin MA, Elemento O, Pestova TV, Qian SB, Jaffrey SR (2015) 5' UTR m(6)A promotes cap-independent translation. Cell 163:999–1010CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Hussain S, Bashir ZI (2015) The epitranscriptome in modulating spatiotemporal RNA translation in neuronal post-synaptic function. Front Cell Neurosci 9:420CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Bokar JA, Shambaugh ME, Polayes D, Matera AG, Rottman FM (1997) Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase. RNA 3:1233–1247PubMedPubMedCentralGoogle Scholar
  12. 12.
    Jia G, Fu Y, Zhao X, Dai Q, Zheng G, Yang Y et al (2011) N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol 7:885–887CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Ule J, Jensen KB, Ruggiu M, Mele A, Ule A, Darnell RB (2003) CLIP identifiesNova-regulated RNA networks in the brain. Science 302:1212–1215CrossRefPubMedGoogle Scholar
  14. 14.
    Licatalosi DD, Mele A, Fak JJ, Ule J, Kayikci M, Chi SW, Clark TA, Schweitzer AC, Blume JE, Wang X, Darnell JC, Darnell RB (2008) HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456:464–469CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Zhang C, Darnell RB (2011) Mapping in vivo protein-RNA interactions at single-nucleotide resolution from HITS-CLIP data. Nat Biotechnol 29:607–614CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Sugimoto Y, Konig J, Hussain S, Zupan B, Curk T, Frye M, Ule J (2012) Genome Biol 13:R67CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    König J, Zarnack K, Rot G, Curk T, Kayikci M, Zupan B, Turner DJ, Luscombe NM, Ule J (2010) iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol 17:909–915CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    King MY, Redman KL (2002) RNA methyltransferases utilize two cysteine residues in the formation of 5-methylcytosine. Biochemistry 41:11218–11225CrossRefPubMedGoogle Scholar
  19. 19.
    Redman KL (2006) Assembly of protein-RNA complexes using natural RNA and mutant forms of an RNA cytosine methyltransferase. Biomacromolecules 7:3321–3326CrossRefPubMedGoogle Scholar
  20. 20.
    Hussain S, Benavente SB, Nascimento E, Dragoni I, Kurowski A, Gillich A, Humphreys P, Frye M (2009) The nucleolar RNA methyltransferase Misu (NSun2) is required for mitotic spindle stability. J Cell Biol 186:27–40CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Linder B, Grozhik AV, Olarerin-George AO, Meydan C, Mason CE, Jaffrey SR (2015) Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat Methods 12:767–772CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Carlile TM, Rojas-Duran MF, Zinshteyn B, Shin H, Bartoli KM, Gilbert WV (2014) Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells. Nature 515:143–146CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Schwartz S, Bernstein DA, Mumbach MR, Jovanovic M, Herbst RH, León-Ricardo BX et al (2014) Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA. Cell 159:148–162CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Saletore Y, Meyer K, Korlach J, Vilfan ID, Jaffrey S, Mason CE (2012) The birth of the epitranscriptome: deciphering the function of RNA modifications. Genome Biol 13:175CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Hussain S, Aleksic J, Blanco S, Dietmann S, Frye M (2013b) Characterizing 5-methylcytosine in the mammalian epitranscriptome. Genome Biol 14:215CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Rinn JL, Ule J (2014) Oming in on RNA–protein interactions. Genome Biol 15:401CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Chen B, Yun J, Kim MS, Mendell JT, Xie Y (2014) PIPE-CLIP: a comprehensive online tool for CLIP-seq data analysis. Genome Biol 15:R18CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Guelorget A, Golinelli-Pimpaneau B (2011) Mechanism-based strategies for trapping and crystallizing complexes of RNA-modifying enzymes. Structure 19:282–291CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Biology and BiochemistryUniversity of BathBathUK
  2. 2.Department of Molecular NeuroscienceUniversity College LondonLondonUK

Personalised recommendations