Skip to main content
SpringerLink
Log in

Analysis of Nucleoporin Function Using Inducible Degron Techniques

  • Protocol
  • First Online:
The Nuclear Pore Complex

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2502))

Abstract

Over the last decade, the use of auxin-inducible degrons (AID) to control the stability of target proteins has revolutionized the field of cell biology. AID-mediated degradation helps to overcome multiple hurdles that have been encountered in studying multisubunit protein complexes, like the nuclear pore complex (NPC), using classical biochemical and genetic methods. We have used the AID system for acute depletion of individual members of the NPC, called nucleoporins, in order to distinguish their roles both within established NPCs and during NPC assembly.

Here, we describe a protocol for CRISPR/Cas9-mediated gene targeting of genes with the AID tag. As an example, we describe a step-by-step protocol for targeting of the NUP153 gene. We also provide recommendations for screening strategies and integration of the sequence encoding the Transport Inhibitor Response 1 (TIR1) protein, a E3-Ubiquitin ligase subunit necessary for AID-dependent protein degradation. In addition, we discuss applications of the NUP-AID system and functional assays for analysis of NUP-AID tagged cell lines.

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 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 299.99
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

Similar content being viewed by others

Abbreviations

AID:

Auxin-Induced Degron

GOI:

Gene of Interest

gRNA:

guide RNA

HDR:

Homology Directed Repair

NE:

Nuclear Envelope

NPC:

Nuclear Pore Complex

NUP:

Nucleoporin/NPC constituent protein

NUP-AID:

Nucleoporin-AID fusion protein

POI:

Protein of Interest

TIR1:

Transport Inhibitor Response 1 protein/ubiquitin ligase subunit

References

  1. Hampoelz B, Andres-Pons A, Kastritis P, Beck M (2019) Structure and assembly of the nuclear pore complex. Annu Rev Biophys 48:515–536. https://doi.org/10.1146/annurev-biophys-052118-115308

    Article  CAS  PubMed  Google Scholar 

  2. Sakuma S, D'Angelo MA (2017) The roles of the nuclear pore complex in cellular dysfunction, aging and disease. Semin Cell Dev Biol 68:72–84. https://doi.org/10.1016/j.semcdb.2017.05.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Toyama BH, Savas JN, Park SK, Harris MS, Ingolia NT, Yates JR, Hetzer MW (2013) Identification of long-lived proteins reveals exceptional stability of essential cellular structures. Cell 154(5):971–982. https://doi.org/10.1016/j.cell.2013.07.037

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Walther TC, Alves A, Pickersgill H, Loiodice I, Hetzer M, Galy V, Hulsmann BB, Kocher T, Wilm M, Allen T, Mattaj IW, Doye V (2003) The conserved Nup107-160 complex is critical for nuclear pore complex assembly. Cell 113(2):195–206. https://doi.org/10.1016/s0092-8674(03)00235-6

    Article  CAS  PubMed  Google Scholar 

  5. Nishimura K, Fukagawa T, Takisawa H, Kakimoto T, Kanemaki M (2009) An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat Methods 6(12):917–922. https://doi.org/10.1038/nmeth.1401

    Article  CAS  PubMed  Google Scholar 

  6. Hoffmann S, Fachinetti D (2018) Real-time De novo deposition of centromeric histone-associated proteins using the auxin-inducible degradation system. Methods Mol Biol 1832:223–241. https://doi.org/10.1007/978-1-4939-8663-7_12

    Article  CAS  PubMed  Google Scholar 

  7. Shetty A, Reim NI, Winston F (2019) Auxin-inducible Degron system for depletion of proteins in Saccharomyces cerevisiae. Curr Protoc Mol Biol 128(1):e104. https://doi.org/10.1002/cpmb.104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Nishimura K, Kanemaki MT (2014) Rapid depletion of budding yeast proteins via the fusion of an auxin-inducible Degron (AID). Curr Protoc Cell Biol 64:20.9.1–20.916. https://doi.org/10.1002/0471143030.cb2009s64

    Article  Google Scholar 

  9. Zhang L, Ward JD, Cheng Z, Dernburg AF (2015) The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans. Development 142(24):4374–4384. https://doi.org/10.1242/dev.129635

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bence M, Jankovics F, Lukacsovich T, Erdelyi M (2017) Combining the auxin-inducible degradation system with CRISPR/Cas9-based genome editing for the conditional depletion of endogenous Drosophila melanogaster proteins. FEBS J 284(7):1056–1069. https://doi.org/10.1111/febs.14042

    Article  CAS  PubMed  Google Scholar 

  11. Nielsen CF, Zhang T, Barisic M, Kalitsis P, Hudson DF (2020) Topoisomerase IIalpha is essential for maintenance of mitotic chromosome structure. Proc Natl Acad Sci U S A 117(22):12131–12142. https://doi.org/10.1073/pnas.2001760117

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Sathyan KM, McKenna BD, Anderson WD, Duarte FM, Core L, Guertin MJ (2019) An improved auxin-inducible degron system preserves native protein levels and enables rapid and specific protein depletion. Genes Dev 33(19–20):1441–1455. https://doi.org/10.1101/gad.328237.119

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Nora EP, Goloborodko A, Valton AL, Gibcus JH, Uebersohn A, Abdennur N, Dekker J, Mirny LA, Bruneau BG (2017) Targeted degradation of CTCF decouples local insulation of chromosome domains from genomic compartmentalization. Cell 169(5):930–944.e22. https://doi.org/10.1016/j.cell.2017.05.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lambrus BG, Moyer TC, Holland AJ (2018) Applying the auxin-inducible degradation system for rapid protein depletion in mammalian cells. Methods Cell Biol 144:107–135. https://doi.org/10.1016/bs.mcb.2018.03.004

    Article  CAS  PubMed  Google Scholar 

  15. Holland AJ, Fachinetti D, Han JS, Cleveland DW (2012) Inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells. Proc Natl Acad Sci U S A 109(49):E3350–E3357. https://doi.org/10.1073/pnas.1216880109

    Article  PubMed  PubMed Central  Google Scholar 

  16. Boer J, Bonten-Surtel J, Grosveld G (1998) Overexpression of the nucleoporin CAN/NUP214 induces growth arrest, nucleocytoplasmic transport defects, and apoptosis. Mol Cell Biol 18(3):1236–1247. https://doi.org/10.1128/mcb.18.3.1236

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Clift D, So C, McEwan WA, James LC, Schuh M (2018) Acute and rapid degradation of endogenous proteins by trim-away. Nat Protoc 13(10):2149–2175. https://doi.org/10.1038/s41596-018-0028-3

    Article  CAS  PubMed  Google Scholar 

  18. Natsume T, Kiyomitsu T, Saga Y, Kanemaki MT (2016) Rapid protein depletion in human cells by auxin-inducible degron tagging with short homology donors. Cell Rep 15(1):210–218. https://doi.org/10.1016/j.celrep.2016.03.001

    Article  CAS  PubMed  Google Scholar 

  19. Sathyan KM, Scott TG, Guertin MJ (2020) ARF-AID: a rapidly inducible protein degradation system that preserves basal endogenous protein levels. Curr Protoc Mol Biol 132(1):e124. https://doi.org/10.1002/cpmb.124

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Yesbolatova A, Saito Y, Kitamoto N, Makino-Itou H, Ajima R, Nakano R, Nakaoka H, Fukui K, Gamo K, Tominari Y, Takeuchi H, Saga Y, Hayashi KI, Kanemaki MT (2020) The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice. Nat Commun 11(1):5701. https://doi.org/10.1038/s41467-020-19532-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lin DH, Hoelz A (2019) The structure of the nuclear pore complex (an update). Annu Rev Biochem 88:725–783. https://doi.org/10.1146/annurev-biochem-062917-011901

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Hu JH, Miller SM, Geurts MH, Tang W, Chen L, Sun N, Zeina CM, Gao X, Rees HA, Lin Z, Liu DR (2018) Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556(7699):57–63. https://doi.org/10.1038/nature26155

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kim HK, Lee S, Kim Y, Park J, Min S, Choi JW, Huang TP, Yoon S, Liu DR, Kim HH (2020) High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells. Nat Biomed Eng 4(1):111–124. https://doi.org/10.1038/s41551-019-0505-1

    Article  CAS  PubMed  Google Scholar 

  24. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823. https://doi.org/10.1126/science.1231143

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Quax TE, Claassens NJ, Soll D, van der Oost J (2015) Codon bias as a means to fine-tune gene expression. Mol Cell 59(2):149–161. https://doi.org/10.1016/j.molcel.2015.05.035

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Chen X, Zaro JL, Shen WC (2013) Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev 65(10):1357–1369. https://doi.org/10.1016/j.addr.2012.09.039

    Article  CAS  PubMed  Google Scholar 

  27. Blancher C, Jones A (2001) SDS -PAGE and Western blotting techniques. Methods Mol Med 57:145–162. https://doi.org/10.1385/1-59259-136-1:145

    Article  CAS  PubMed  Google Scholar 

  28. Aksenova V, Smith A, Lee H, Bhat P, Esnault C, Chen S, Iben J, Kaufhold R, Yau KC, Echeverria C, Fontoura B, Arnaoutov A, Dasso M (2020) Nucleoporin TPR is an integral component of the TREX-2 mRNA export pathway. Nat Commun 11(1):4577. https://doi.org/10.1038/s41467-020-18266-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Yau KC, Arnaoutov A, Aksenova V, Kaufhold R, Chen S, Dasso M (2020) RanBP1 controls the Ran pathway in mammalian cells through regulation of mitotic RCC1 dynamics. Cell Cycle 19(15):1899–1916. https://doi.org/10.1080/15384101.2020.1782036

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Morawska M, Ulrich HD (2013) An expanded tool kit for the auxin-inducible degron system in budding yeast. Yeast 30(9):341–351. https://doi.org/10.1002/yea.2967

    Article  CAS  PubMed  Google Scholar 

  31. Feoktistova M, Geserick P, Leverkus M (2016) Crystal violet assay for determining viability of cultured cells. Cold Spring Harb Protoc 2016(4):pdb.prot087379. https://doi.org/10.1101/pdb.prot087379

    Article  PubMed  Google Scholar 

  32. Niopek D, Wehler P, Roensch J, Eils R, Di Ventura B (2016) Optogenetic control of nuclear protein export. Nat Commun 7:10624. https://doi.org/10.1038/ncomms10624

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK (2014) Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol 32(3):279–284. https://doi.org/10.1038/nbt.2808

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Tan X, Calderon-Villalobos LI, Sharon M, Zheng C, Robinson CV, Estelle M, Zheng N (2007) Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature 446(7136):640–645. https://doi.org/10.1038/nature05731

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development at the National Institutes of Health, USA (Intramural Project #Z01 HD008954). We thank Ashley Person, NIH postbaccalaureate fellow, for comments on this manuscript.

Author information

Authors and Affiliations

  1. Division of Molecular and Cellular Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA

    Vasilisa Aksenova, Alexei Arnaoutov & Mary Dasso

Authors
  1. Vasilisa Aksenova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alexei Arnaoutov
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mary Dasso
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Vasilisa Aksenova .

Editor information

Editors and Affiliations

  1. Department of Biosciences, Durham University, Durham, UK

    Martin W. Goldberg

Rights and permissions

Reprints and permissions

Copyright information

© 2022 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Aksenova, V., Arnaoutov, A., Dasso, M. (2022). Analysis of Nucleoporin Function Using Inducible Degron Techniques. In: Goldberg, M.W. (eds) The Nuclear Pore Complex. Methods in Molecular Biology, vol 2502. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2337-4_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2337-4_9

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2336-7

  • Online ISBN: 978-1-0716-2337-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation