Skip to main content
SpringerLink
Log in

Identifying miRNA Targets Using AGO-RIPseq

  • Protocol
  • First Online:
mRNA Decay

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1720))

Abstract

microRNAs (miRNA) are small, noncoding RNAs that bind to messenger RNAs (mRNAs) and regulate their activity. They are, therefore, important posttranscriptional regulators. In recent years it has become clear that miRNAs regulate large genetic networks, rather than single genes, and that one gene can be targeted by several miRNAs. To understand the role of miRNAs in cells or tissues, it is therefore important to analyze the targetome of miRNAs. Here, we present a technique called Argonaute-RNA Immunoprecipitation (AGO-RIP) which takes advantages of the fact that miRNAs and their targets are directly bound by the Argonaute protein family. With this approach quantitative, genome-wide analysis of miRNA targets is possible. In this chapter we describe the RIP-methodology and provide advice for RNA sequencing and bioinformatic analyses.

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

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136(2):215–233. https://doi.org/10.1016/j.cell.2009.01.002. S0092-8674(09)00008-7 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281–297

    Article  CAS  PubMed  Google Scholar 

  3. Krol J, Loedige I, Filipowicz W (2010) The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet 11(9):597–610. https://doi.org/10.1038/nrg2843

    CAS  PubMed  Google Scholar 

  4. Kim VN (2005) MicroRNA biogenesis: coordinated cropping and dicing. Nat Rev Mol Cell Biol 6(5):376–385. https://doi.org/10.1038/nrm1644

    Article  CAS  PubMed  Google Scholar 

  5. Ebert MS, Sharp PA (2012) Roles for microRNAs in conferring robustness to biological processes. Cell 149(3):515–524. https://doi.org/10.1016/j.cell.2012.04.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Guo H, Ingolia NT, Weissman JS, Bartel DP (2010) Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466(7308):835–840. https://doi.org/10.1038/nature09267

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hammell M, Long D, Zhang L, Lee A, Carmack CS, Han M, Ding Y, Ambros V (2008) mirWIP: microRNA target prediction based on microRNA-containing ribonucleoprotein-enriched transcripts. Nat Methods 5(9):813–819. https://doi.org/10.1038/nmeth.1247

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Ritchie W, Flamant S, Rasko JE (2009) Predicting microRNA targets and functions: traps for the unwary. Nat Methods 6(6):397–398. https://doi.org/10.1038/nmeth0609-397

    Article  CAS  PubMed  Google Scholar 

  9. Boudreau RL, Jiang P, Gilmore BL, Spengler RM, Tirabassi R, Nelson JA, Ross CA, Xing Y, Davidson BL (2014) Transcriptome-wide discovery of microRNA binding sites in human brain. Neuron 81(2):294–305. https://doi.org/10.1016/j.neuron.2013.10.062

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Chi SW, Zang JB, Mele A, Darnell RB (2009) Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460(7254):479–486. https://doi.org/10.1038/nature08170

    CAS  PubMed  PubMed Central  Google Scholar 

  11. He M, Liu Y, Wang X, Zhang MQ, Hannon GJ, Huang ZJ (2012) Cell-type-based analysis of microRNA profiles in the mouse brain. Neuron 73(1):35–48. https://doi.org/10.1016/j.neuron.2011.11.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Helwak A, Tollervey D (2014) Mapping the miRNA interactome by cross-linking ligation and sequencing of hybrids (CLASH). Nat Protoc 9(3):711–728. https://doi.org/10.1038/nprot.2014.043

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Malmevik J, Petri R, Klussendorf T, Knauff P, Akerblom M, Johansson J, Soneji S, Jakobsson J (2015) Identification of the miRNA targetome in hippocampal neurons using RIP-seq. Sci Rep 5:12609. https://doi.org/10.1038/srep12609

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Petri R, Pircs K, Jonsson ME, Akerblom M, Brattas PL, Klussendorf T, Jakobsson J (2017) let-7 regulates radial migration of new-born neurons through positive regulation of autophagy. EMBO J. 10.15252/embj.201695235

    Google Scholar 

  15. Hammond SM, Boettcher S, Caudy AA, Kobayashi R, Hannon GJ (2001) Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293(5532):1146–1150. https://doi.org/10.1126/science.1064023

    Article  CAS  PubMed  Google Scholar 

  16. Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 17(1):10–12

    Article  Google Scholar 

  17. 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 

  18. Liao Y, Smyth GK, Shi W (2014) featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30(7):923–930. https://doi.org/10.1093/bioinformatics/btt656

    Article  CAS  PubMed  Google Scholar 

  19. Kozomara A, Griffiths-Jones S (2014) miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res 42(Database issue):D68–D73. https://doi.org/10.1093/nar/gkt1181

    Article  CAS  PubMed  Google Scholar 

  20. Kersey PJ, Allen JE, Armean I, Boddu S, Bolt BJ, Carvalho-Silva D, Christensen M, Davis P, Falin LJ, Grabmueller C, Humphrey J, Kerhornou A, Khobova J, Aranganathan NK, Langridge N, Lowy E, McDowall MD, Maheswari U, Nuhn M, Ong CK, Overduin B, Paulini M, Pedro H, Perry E, Spudich G, Tapanari E, Walts B, Williams G, Tello-Ruiz M, Stein J, Wei S, Ware D, Bolser DM, Howe KL, Kulesha E, Lawson D, Maslen G, Staines DM (2016) Ensembl Genomes 2016: more genomes, more complexity. Nucleic Acids Res 44(D1):D574–D580. https://doi.org/10.1093/nar/gkv1209

    Article  CAS  PubMed  Google Scholar 

  21. Rosenbloom KR, Armstrong J, Barber GP, Casper J, Clawson H, Diekhans M, Dreszer TR, Fujita PA, Guruvadoo L, Haeussler M, Harte RA, Heitner S, Hickey G, Hinrichs AS, Hubley R, Karolchik D, Learned K, Lee BT, Li CH, Miga KH, Nguyen N, Paten B, Raney BJ, Smit AF, Speir ML, Zweig AS, Haussler D, Kuhn RM, Kent WJ (2015) The UCSC Genome Browser database: 2015 update. Nucleic Acids Res 43(Database issue):D670–D681. https://doi.org/10.1093/nar/gku1177

    Article  CAS  PubMed  Google Scholar 

  22. O’Leary NA, Wright MW, Brister JR, Ciufo S, Haddad D, McVeigh R, Rajput B, Robbertse B, Smith-White B, Ako-Adjei D, Astashyn A, Badretdin A, Bao Y, Blinkova O, Brover V, Chetvernin V, Choi J, Cox E, Ermolaeva O, Farrell CM, Goldfarb T, Gupta T, Haft D, Hatcher E, Hlavina W, Joardar VS, Kodali VK, Li W, Maglott D, Masterson P, McGarvey KM, Murphy MR, O’Neill K, Pujar S, Rangwala SH, Rausch D, Riddick LD, Schoch C, Shkeda A, Storz SS, Sun H, Thibaud-Nissen F, Tolstoy I, Tully RE, Vatsan AR, Wallin C, Webb D, Wu W, Landrum MJ, Kimchi A, Tatusova T, DiCuccio M, Kitts P, Murphy TD, Pruitt KD (2016) Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res 44(D1):D733–D745. https://doi.org/10.1093/nar/gkv1189

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratory of Molecular Neurogenetics, Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, Lund University, Lund, Sweden

    Rebecca Petri & Johan Jakobsson

Authors
  1. Rebecca Petri
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Johan Jakobsson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Johan Jakobsson .

Editor information

Editors and Affiliations

  1. Murdoch Children’s Research Institute, The Royal Children’s Hospital, Parkville, Victoria, Australia

    Shireen R. Lamandé

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Petri, R., Jakobsson, J. (2018). Identifying miRNA Targets Using AGO-RIPseq. In: Lamandé, S. (eds) mRNA Decay. Methods in Molecular Biology, vol 1720. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7540-2_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7540-2_9

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7539-6

  • Online ISBN: 978-1-4939-7540-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation