Skip to main content
SpringerLink
Log in

HAMR: High-Throughput Annotation of Modified Ribonucleotides

  • Protocol
  • First Online:
Epitranscriptomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1870))

Abstract

Ribonucleotides can be decorated with over 100 types of covalent chemical modifications. These modifications change the structure, function, and catalytic activity of RNAs, forming a layer of posttranscriptional regulation termed the epitranscriptome. Recent advances in high-throughput mapping have demonstrated these modifications are abundant and mark nearly all classes of RNAs, including messenger RNAs. Here, we outline one such technique called high-throughput annotation of modified ribonucleotides (HAMR). HAMR exploits the tendency of certain modified ribonucleotides to interfere with base pairing, leading to errors in complementary DNA synthesis during RNA sequencing library preparation. In total, we present a computational protocol for in silico identification of modifications with HAMR, which can be retroactively applied to a variety of RNA sequencing techniques.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  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. https://doi.org/10.1093/nar/gks1007

    Article  CAS  PubMed  Google Scholar 

  2. Dunin-Horkawicz S, Czerwoniec A, Gajda MJ, Feder M, Grosjean H, Bujnicki JM (2006) MODOMICS: a database of RNA modification pathways. Nucleic Acids Res 34(Database issue):D145–D149. https://doi.org/10.1093/nar/gkj084

    Article  CAS  PubMed  Google Scholar 

  3. Limbach PA, Crain PF, McCloskey JA (1994) Summary: the modified nucleosides of RNA. Nucleic Acids Res 22(12):2183–2196

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Cantara WA, Crain PF, Rozenski J, McCloskey JA, Harris KA, Zhang X, Vendeix FA, Fabris D, Agris PF (2011) The RNA modification database, RNAMDB: 2011 update. Nucleic Acids Res 39(Database):D195–D201. https://doi.org/10.1093/nar/gkq1028

    Article  CAS  PubMed  Google Scholar 

  5. Helm M, Giege R, Florentz C (1999) A Watson-crick base-pair-disrupting methyl group (m1A9) is sufficient for cloverleaf folding of human mitochondrial tRNALys. Biochemistry 38(40):13338–13346

    Article  CAS  PubMed  Google Scholar 

  6. Sundaram M, Durant PC, Davis DR (2000) Hypermodified nucleosides in the anticodon of tRNALys stabilize a canonical U-turn structure. Biochemistry 39(41):12575–12584

    Article  CAS  PubMed  Google Scholar 

  7. Patil DP, Chen CK, Pickering BF, Chow A, Jackson C, Guttman M, Jaffrey SR (2016) M(6)a RNA methylation promotes XIST-mediated transcriptional repression. Nature 537(7620):369–373. https://doi.org/10.1038/nature19342

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Zhao X, Yang Y, Sun BF, Shi Y, Yang X, Xiao W, Hao YJ, Ping XL, Chen YS, Wang WJ, Jin KX, Wang X, Huang CM, Fu Y, Ge XM, Song SH, Jeong HS, Yanagisawa H, Niu Y, Jia GF, Wu W, Tong WM, Okamoto A, He C, Rendtlew Danielsen JM, Wang XJ, Yang YG (2014) FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis. Cell Res 24(12):1403–1419. https://doi.org/10.1038/cr.2014.151

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Xiao W, Adhikari S, Dahal U, Chen YS, Hao YJ, Sun BF, Sun HY, Li A, Ping XL, Lai WY, Wang X, Ma HL, Huang CM, Yang Y, Huang N, Jiang GB, Wang HL, Zhou Q, Wang XJ, Zhao YL, Yang YG (2016) Nuclear m(6)A reader YTHDC1 regulates mRNA splicing. Mol Cell 61(4):507–519. https://doi.org/10.1016/j.molcel.2016.01.012

    Article  CAS  PubMed  Google Scholar 

  10. Wang X, Zhao BS, Roundtree IA, Lu Z, Han D, Ma H, Weng X, Chen K, Shi H, He C (2015) N(6)-methyladenosine modulates messenger RNA translation efficiency. Cell 161(6):1388–1399. https://doi.org/10.1016/j.cell.2015.05.014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Choi J, Ieong KW, Demirci H, Chen J, Petrov A, Prabhakar A, O'Leary SE, Dominissini D, Rechavi G, Soltis SM, Ehrenberg M, Puglisi JD (2016) N(6)-methyladenosine in mRNA disrupts tRNA selection and translation-elongation dynamics. Nat Struct Mol Biol 23(2):110–115. https://doi.org/10.1038/nsmb.3148

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Du H, Zhao Y, He J, Zhang Y, Xi H, Liu M, Ma J, Wu L (2016) YTHDF2 destabilizes m(6)A-containing RNA through direct recruitment of the CCR4-NOT deadenylase complex. Nat Commun 7:12626. https://doi.org/10.1038/ncomms12626

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wang Y, Li Y, Toth JI, Petroski MD, Zhang Z, Zhao JC (2014) N6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells. Nat Cell Biol 16(2):191–198. https://doi.org/10.1038/ncb2902

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wang X, Lu Z, Gomez A, Hon GC, Yue Y, Han D, Fu Y, Parisien M, Dai Q, Jia G, Ren B, Pan T, He C (2014) N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505(7481):117–120. https://doi.org/10.1038/nature12730

    Article  CAS  PubMed  Google Scholar 

  15. Mauer J, Luo X, Blanjoie A, Jiao X, Grozhik AV, Patil DP, Linder B, Pickering BF, Vasseur JJ, Chen Q, Gross SS, Elemento O, Debart F, Kiledjian M, Jaffrey SR (2017) Reversible methylation of m(6)Am in the 5′ cap controls mRNA stability. Nature 541(7637):371–375. https://doi.org/10.1038/nature21022

    Article  CAS  PubMed  Google Scholar 

  16. Vandivier LE, Gregory BD (2017) Reading the epitranscriptome: new techniques and perspectives enzymes. Enzymes 41:269–298. https://doi.org/10.1016/bs.enz.2017.03.004

    Article  PubMed  Google Scholar 

  17. Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S, Cesarkas K, Jacob-Hirsch J, Amariglio N, Kupiec M, Sorek R, Rechavi G (2012) Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485(7397):201–206. https://doi.org/10.1038/nature11112

    Article  CAS  PubMed  Google Scholar 

  18. Schwartz S, Mumbach MR, Jovanovic M, Wang T, Maciag K, Bushkin GG, Mertins P, Ter-Ovanesyan D, Habib N, Cacchiarelli D, Sanjana NE, Freinkman E, Pacold ME, Satija R, Mikkelsen TS, Hacohen N, Zhang F, Carr SA, Lander ES, Regev A (2014) Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5′ sites. Cell Rep 8(1):284–296. https://doi.org/10.1016/j.celrep.2014.05.048

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 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(7525):143–146. https://doi.org/10.1038/nature13802

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Li X, Zhu P, Ma S, Song J, Bai J, Sun F, Yi C (2015) Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome. Nat Chem Biol 11(8):592–597. https://doi.org/10.1038/nchembio.1836

    Article  CAS  PubMed  Google Scholar 

  21. Schwartz S, Bernstein DA, Mumbach MR, Jovanovic M, Herbst RH, Leon-Ricardo BX, Engreitz JM, Guttman M, Satija R, Lander ES, Fink G, Regev A (2014) Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA. Cell 159(1):148–162. https://doi.org/10.1016/j.cell.2014.08.028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Lovejoy AF, Riordan DP, Brown PO (2014) Transcriptome-wide mapping of pseudouridines: pseudouridine synthases modify specific mRNAs in S. cerevisiae. PLoS One 9(10):e110799. https://doi.org/10.1371/journal.pone.0110799

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Dominissini D, Nachtergaele S, Moshitch-Moshkovitz S, Peer E, Kol N, Ben-Haim MS, Dai Q, Di Segni A, Salmon-Divon M, Clark WC, Zheng G, Pan T, Solomon O, Eyal E, Hershkovitz V, Han D, Dore LC, Amariglio N, Rechavi G, He C (2016) The dynamic N(1)-methyladenosine methylome in eukaryotic messenger RNA. Nature 530(7591):441–446. https://doi.org/10.1038/nature16998

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Li X, Xiong X, Wang K, Wang L, Shu X, Ma S, Yi C (2016) Transcriptome-wide mapping reveals reversible and dynamic N(1)-methyladenosine methylome. Nat Chem Biol 12(5):311–316. https://doi.org/10.1038/nchembio.2040

    Article  CAS  PubMed  Google Scholar 

  25. Ryvkin P, Leung YY, Silverman IM, Childress M, Valladares O, Dragomir I, Gregory BD, Wang LS (2013) HAMR: high-throughput annotation of modified ribonucleotides. RNA 19(12):1684–1692. https://doi.org/10.1261/rna.036806.112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R (2009) The sequence alignment/map format and SAMtools. Bioinformatics. Genome Project Data Processing Subgroup 25(16):2078–2079. https://doi.org/10.1093/bioinformatics/btp352

    Article  CAS  Google Scholar 

  27. Quinlan AR, Hall IM (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26(6):841–842. https://doi.org/10.1093/bioinformatics/btq033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnetjournal 17:10–12

    Google Scholar 

  29. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29(1):15–21. https://doi.org/10.1093/bioinformatics/bts635

    Article  CAS  PubMed  Google Scholar 

  30. Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25(9):1105–1111. https://doi.org/10.1093/bioinformatics/btp120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 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(4):R36. https://doi.org/10.1186/gb-2013-14-4-r36

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA (2010) The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20(9):1297–1303. https://doi.org/10.1101/gr.107524.110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Lowe TM, Chan PP (2016) tRNAscan-SE On-line: integrating search and context for analysis of transfer RNA genes. Nucleic Acids Res 44(W1):W54–W57. https://doi.org/10.1093/nar/gkw413

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Vandivier LE, Campos R, Kuksa PP, Silverman IM, Wang LS, Gregory BD (2015) Chemical modifications mark alternatively spliced and uncapped messenger RNAs in Arabidopsis. Plant Cell 27(11):3024–3037. https://doi.org/10.1105/tpc.15.00591

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Willmann MR, Berkowitz ND, Gregory BD (2014) Improved genome-wide mapping of uncapped and cleaved transcripts in eukaryotes—GMUCT 2.0. Methods 67(1):64–73. https://doi.org/10.1016/j.ymeth.2013.07.003

    Article  CAS  PubMed  Google Scholar 

  36. Gregory BD, O’Malley RC, Lister R, Urich MA, Tonti-Filippini J, Chen H, Millar AH, Ecker JR (2008) A link between RNA metabolism and silencing affecting Arabidopsis development. Dev Cell 14(6):854–866. https://doi.org/10.1016/j.devcel.2008.04.005

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors would like to members of the Gregory and Wang labs both past and present for helpful discussions. This work was funded by NSF grants MCB-1623887 and IOS-1444490 to B.D.G.

Author information

Author notes

  1. Lee E. Vandivier and Zachary D. Anderson contributed equally to this work

Authors and Affiliations

  1. Department of Biology, University of Pennsylvania, Philadelphia, PA, USA

    Lee E. Vandivier, Zachary D. Anderson & Brian D. Gregory

  2. Cell and Molecular Biology Graduate Program, University of Pennsylvania, Philadelphia, PA, USA

    Lee E. Vandivier & Brian D. Gregory

Authors
  1. Lee E. Vandivier
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zachary D. Anderson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Brian D. Gregory
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Brian D. Gregory .

Editor information

Editors and Affiliations

  1. Department of Pathology, Yale University School of Medicine, New Haven, CT, USA

    Narendra Wajapeyee

  2. Yale Cancer Center, Yale University School of Medicine, New Haven, CT, USA

    Romi Gupta

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Vandivier, L.E., Anderson, Z.D., Gregory, B.D. (2019). HAMR: High-Throughput Annotation of Modified Ribonucleotides. In: Wajapeyee, N., Gupta, R. (eds) Epitranscriptomics. Methods in Molecular Biology, vol 1870. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8808-2_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8808-2_4

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8807-5

  • Online ISBN: 978-1-4939-8808-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation