Crosslinking Methods to Identify RNA Methyltransferase Targets In Vivo

  • Sara Haag
  • Jens Kretschmer
  • Katherine E. Sloan
  • Markus T. BohnsackEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1562)


Several crosslinking methods have been developed to identify interacting RNAs for proteins of interest. Here, we describe variants of the UV crosslinking and analysis of cDNA (CRAC) method that allow target identification of RNA methyltransferases on a genome-wide scale. We present a detailed protocol for the application of CRAC in human cells that stably express the protein of interest fused to a tandem affinity tag. After the introduction of a covalent link between the protein and its target RNAs, protein-RNA complexes are purified and bound RNAs trimmed, ligated to adapters, reverse transcribed, and amplified. Sequences obtained from next-generation sequencing are then mapped onto the human genome allowing the identification of possible substrates. For some RNA methyltransferases, e.g., m5C MTases, their catalytic mechanism can be exploited for chemical crosslinking approaches instead of UV based crosslinking.

Key words

UV crosslinking CRAC Methyltransferase CLIP 5-Methylcytosine 5-Azacytidine 



This work was supported by the Deutsche Forschungsgemeinschaft (SPP 1784: BO3442/2-1 to M.T.B.), the Alexander von Humboldt Foundation (K.E.S. and M.T.B.), and the Faculty of Medicine, Georg-August-University Göttingen (M.T.B. and “Startförderung” to S.H.).


  1. 1.
    Petrossian T, Clarke S (2009) Bioinformatic identification of novel methyltransferases. Epigenomics 1:163–175CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Petrossian TC, Clarke SG (2011) Uncovering the human methyltransferasome. Mol Cell Proteomics 10:M110.000976CrossRefPubMedGoogle Scholar
  3. 3.
    Ule J, Jensen KB, Ruggiu M, Mele A, Ule A, Darnell RB (2003) CLIP identifies Nova-regulated RNA networks in the brain. Science 302:1212–1215CrossRefPubMedGoogle Scholar
  4. 4.
    Licatalosi DD, Mele A, Fak JJ, Ule J, Kayikci M, Chi SW et al (2008) HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456:464–469CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Zhang C, Darnell RB (2011) Mapping in vivo protein-RNA interactions at single-nucleotide resolution from HITS-CLIP data. NatBiotechnol 29:607–614Google Scholar
  6. 6.
    Wang Z, Kayikci M, Briese M, Zarnack K, Luscombe NM, Rot G et al (2010) iCLIP predicts the dual splicing effects of TIA-RNA interactions. PLoSBiol 8:e1000530CrossRefGoogle Scholar
  7. 7.
    Konig J, Zarnack K, Rot G, Curk T, Kayikci M, Zupan B et al (2010) iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol 17:909–915CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Bohnsack MT, Tollervey D, Granneman S (2012) Identification of RNA helicase target sites by UV cross-linking and analysis of cDNA. Methods Enzymol 511:275–288CrossRefPubMedGoogle Scholar
  9. 9.
    Grannemann S, Kudla G, Petfalski E, Tollervey D (2009) Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high-throughput analysis of cDNAs. Proc Natl Acad Sci U S A 106:9613–9618CrossRefGoogle Scholar
  10. 10.
    Granneman S, Petfalski E, Swiatkowska A, Tollervey D (2010) Cracking pre-40S ribosomal subunit structure by systematic analyses of RNA-protein cross-linking. EMBO J 29:2026–2036CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Bohnsack MT, Martin R, Granneman S, Ruprecht M, Schleiff E, Tollervey D (2009) Prp43 bound at different sites on the pre-rRNA performs distinct functions in ribosome synthesis. Mol Cell 36:583–592CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Martin R, Hackert P, Ruprecht M, Simm S, Brüning L, Mirus O et al (2014) A pre-ribosomal RNA interaction network involving snoRNAs and the Rok1 helicase. RNA 20:1173–1182CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Sloan KE, Leisegang MS, Doebele C, Ramirez AS, Simm S, Safferthal C et al (2015) The association of late-acting snoRNPs with human pre-ribosomal complexes requires the RNA helicase DDX21. Nucleic Acids Res 43, 553–564.Google Scholar
  14. 14.
    Haag S, Warda AS, Kretschmer J, Günnigmann MA, Höbartner C, Bohnsack MT (2015) NSUN6 is a human RNA methyltransferase that catalyzes formation of m5C72 in specific tRNAs. RNA 21:1532–1543CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Hafner M, Landthaler M, Burger L, Khorshid M, Hausser J, Berninger P et al (2010) Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141:129–141CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Liu Y, Santi DV (2000) m5C RNA and m5C DNA methyl transferases use different cysteine residues as catalysts. Proc Natl Acad Sci U S A 97:8263–8265CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    King MY, Redman KL (2002) RNA methyltransferases utilize two cysteine residues in the formation of 5-methylcytosine. Biochemistry 41:11218–11225CrossRefPubMedGoogle Scholar
  18. 18.
    Hussain S, Sajini AA, Blanco S, Dietmann S, Lombard P, Sugimoto Y et al (2013) NSun2-mediated cytosine-5 methylation of vault noncoding RNA determines its processing into regulatory small RNAs. Cell Rep 4:255–261CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Khoddami V, Cairns BR (2013) Identification of direct targets and modified bases of RNA cytosine methyltransferases. Nat Biotechnol 31:458–464CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Haag S, Höbartner C, and Bohnsack MT (2016) In vitro assays for RNA methyltransferase activity. Methods Mol Biol, in this issue.Google Scholar
  21. 21.
    Dodt M, Roehr JT, Ahmed R, Dieterich C (2012) Flexbar - flexible barcode and adapter processing for next-generation sequencing platforms. Biology 1:895–905CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. GenomeBiol 10:R25Google Scholar
  23. 23.
    Langmead B, Salzberg S (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL (2013) TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol 14:R36CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Khorshid M, Rodak C, Zavolan M (2011) CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins. Nucleic Acids Res 39:D245–D252CrossRefPubMedGoogle Scholar
  26. 26.
    Webb S, Hector RD, Kudla G, Granneman S (2014) PAR-CLIP data indicate that Nrd1-Nab3-dependent transcription termination regulates expression of hundreds of protein coding genes in yeast. Genome Biol 15:R8CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Chi SW, Zang JB, Mele A, Darnell RB (2009) Ago HITS-CLIP decodes miRNA-mRNA interaction maps. Nature 460:479–486PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Sara Haag
    • 1
  • Jens Kretschmer
    • 1
  • Katherine E. Sloan
    • 1
  • Markus T. Bohnsack
    • 1
    • 2
    Email author
  1. 1.Centre for Biochemistry and Molecular Cell BiologyInstitute for Molecular Biology, Georg-August-UniversityGöttingenGermany
  2. 2.Göttingen Centre for Molecular BiosciencesGeorg-August-UniversityGöttingenGermany

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