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Native Circular RNA Pulldown Method to Simultaneously Profile RNA and Protein Interactions

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Circular RNAs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2765))

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Abstract

Circular RNAs (circRNAs) are a widespread, cell-, tissue-, and disease-specific class of largely non-coding RNA transcripts. These single-stranded, covalently-closed transcripts arise through non-canonical splicing of pre-mRNA, a process called back-splicing. Back-splicing results in circRNAs which are distinguishable from their cognate mRNA as they possess a unique sequence of nucleic acids called the backsplice junction (BSJ). CircRNAs have been shown to play key functional roles in various cellular contexts and achieve this through their interaction with other macromolecules, particularly other RNA molecules and proteins. To elucidate the molecular mechanisms underlying circRNA function, it is necessary to identify these interacting partners. Herein, we present an optimized strategy for the simultaneous purification of the circRNA interactome within eukaryotic cells, allowing the identification of both circRNA–RNA and circRNA–protein interactions.

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References

  1. Conn S, Pillman K, Toubia J et al (2015) The RNA binding protein quaking regulates formation of circRNAs. Cell 160:1125–1134

    Article  CAS  PubMed  Google Scholar 

  2. Jeck WR, Sharpless NE (2014) Detecting and characterizing circular RNAs. Nat Biotechnol 32:453–461. https://doi.org/10.1038/nbt.2890

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Rybak-Wolf A, Stottmeister C, Glažar P et al (2015) Circular RNAs in the mammalian brain are highly abundant, conserved, and dynamically expressed. Mol Cell 58:870–885. https://doi.org/10.1016/j.molcel.2015.03.027

    Article  CAS  PubMed  Google Scholar 

  4. Jeck WR, Sorrentino JA, Wang K et al (2013) Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA 19:141–157. https://doi.org/10.1261/rna.035667.112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Panda AC (2018) Circular RNAs act as miRNA sponges. Adv Exp Med Biol 1087:67–79. https://doi.org/10.1007/978-981-13-1426-1_6

    Article  CAS  PubMed  Google Scholar 

  6. Hansen TB, Jensen TI, Clausen BH et al (2013) Natural RNA circles function as efficient microRNA sponges. Nature 495:384–388. https://doi.org/10.1038/nature11993

    Article  CAS  PubMed  Google Scholar 

  7. Memczak S, Jens M, Elefsinioti A et al (2013) Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495:333–338. https://doi.org/10.1038/nature11928

    Article  CAS  PubMed  Google Scholar 

  8. Lasda E, Parker R (2014) Circular RNAs: diversity of form and function. RNA 20:1829–1842. https://doi.org/10.1261/rna.047126.114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. You X, Vlatkovic I, Babic A et al (2015) Neural circular RNAs are derived from synaptic genes and regulated by development and plasticity. Nat Neurosci 18:603–610. https://doi.org/10.1038/nn.3975

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Du WW, Yang W, Liu E et al (2016) Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2. Nucleic Acids Res 44:2846–2858. https://doi.org/10.1093/nar/gkw027

    Article  PubMed  PubMed Central  Google Scholar 

  11. Guarnerio J, Bezzi M, Jeong JC et al (2016) Oncogenic role of fusion-circRNAs derived from cancer-associated chromosomal translocations. Cell 165:289–302. https://doi.org/10.1016/j.cell.2016.03.020

    Article  CAS  PubMed  Google Scholar 

  12. Liu X-Y, Zhang Q, Guo J et al (2022) The role of circular RNAs in the drug resistance of cancers. Front Oncol 11

    Google Scholar 

  13. Gabryelska M, Conn SJ (2023) The RNA Interactome in the hallmarks of cancer, vol 14. WIREs RNA In press

    Google Scholar 

  14. Conn VM, Gabryelska M, Toubia J, Kirk K, Gantley L, Powell JA, Cildir G, Marri S, Liu R, Stringer BW, Townley S, Webb ST, LinH, Samaraweera SE, Bailey S, Moore AS, Maybury M, Liu D, Colella AD, Chataway T, Wallington-Gates CT, Walters L, Sibbons J,Selth LA, Tergaonkar V, D'Andrea RJ, Pitson SM, Goodall GJ, Conn SJ (2023) Circular RNAs drive oncogenic chromosomaltranslocations within the MLL recombinome in leukemia. Cancer Cell 41(7):1309–1326

    Google Scholar 

  15. Conn VM, Hugouvieux V, Nayak A, Conos S, Capovilla G, Cildir G, Jourdain A, Tergaonkar V, Schmid M, Zubieta C, Conn SJ (2017) A CircRNA from SEPALLATA3 Regulates Splicing of its Cognate mRNA Through R-loop Formation. Nature Plants 3:17053

    Google Scholar 

  16. Du WW, Zhang C, Yang W et al (2017) Identifying and characterizing circRNA-protein interaction. Theranostics 7:4183–4191. https://doi.org/10.7150/thno.21299

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Huang A, Zheng H, Wu Z et al (2020) Circular RNA-protein interactions: functions, mechanisms, and identification. Theranostics 10:3503–3517. https://doi.org/10.7150/thno.42174

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Ulshöfer CJ, Pfafenrot C, Bindereif A, Schneider T (2021) Methods to study circRNA-protein interactions. Methods 196:36–46. https://doi.org/10.1016/j.ymeth.2021.04.014

    Article  CAS  PubMed  Google Scholar 

  19. Stringer BW, Gabryelska M, Marri S, Clark L, Lin H, Gantley L, Liu R, Wilusz JE, Conn VM, Conn SJ (2023) Versatiletoolkit for highly-efficient and scarless overexpression of circular RNAs. bioRxiv.11.21.568171. https://doi.org/10.1101/2023.11.21.568171

  20. Liu D, Conn V, Goodall GJ, Conn SJ (2018) A highly efficient strategy for overexpressing circRNAs. Methods Mol Biol 1724:97–105. https://doi.org/10.1007/978-1-4939-7562-4_8

    Article  CAS  PubMed  Google Scholar 

  21. Conn VM, Gabryelska M, Marri S et al (2020) SRRM4 expands the repertoire of circular RNAs by regulating microexon inclusion. Cell 9:2488. https://doi.org/10.3390/cells9112488

    Article  CAS  Google Scholar 

  22. Wang QT, Xiao W, Mindrinos M, Davis RW (2002) Yeast tRNA as carrier in the isolation of microscale RNA for global amplification and expression profiling. Biotechniques 33:788, 790, 792. https://doi.org/10.2144/02334st02

    Article  Google Scholar 

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Acknowledgments

Research reported in this publication was supported by the National Health and Medical Research Council (NHMRC) project grant funding to S.J.C. (GNT1144250). Fellowship support was provided by the Australian Research Council Future Fellowship to S.J.C. (FT160100318) and the NHMRC Investigator Leadership Grant to S.J.C. (GNT1198014). Fellowship support for B.W.S. was provided by the Flinders Foundation Brain Cancer Fellowship. We would like to acknowledge Flinders Proteomics for their guidance and expertise in regards to mass spectrometry methodologies. 

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Authors and Affiliations

  1. Flinders Health and Medical Research Institute (FHMRI), College of Medicine and Public Health, Flinders University, Adelaide, SA, Australia

    Marta M. Gabryelska, Stuart T. Webb, He Lin, Laura Gantley, Kirsty Kirk, Ryan Liu, Brett W. Stringer, Vanessa M. Conn & Simon J. Conn

Authors
  1. Marta M. Gabryelska
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  2. Stuart T. Webb
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  3. He Lin
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  4. Laura Gantley
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  5. Kirsty Kirk
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  6. Ryan Liu
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  7. Brett W. Stringer
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  8. Vanessa M. Conn
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  9. Simon J. Conn
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Corresponding author

Correspondence to Simon J. Conn .

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Editors and Affiliations

  1. Klaus Tschira Institute for Integrative Computational Cardiology, University Heidelberg, Heidelberg, Germany

    Christoph Dieterich

  2. Department of Cellular, Computational and Integrative Biology – CIBIO, University of Trento, Trento, Italy

    Marie-Laure Baudet

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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Gabryelska, M.M. et al. (2024). Native Circular RNA Pulldown Method to Simultaneously Profile RNA and Protein Interactions. In: Dieterich, C., Baudet, ML. (eds) Circular RNAs. Methods in Molecular Biology, vol 2765. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3678-7_16

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  • DOI: https://doi.org/10.1007/978-1-0716-3678-7_16

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3677-0

  • Online ISBN: 978-1-0716-3678-7

  • eBook Packages: Springer Protocols

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