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High-Throughput siRNA Screening Applied to the Ubiquitin–Proteasome System

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Proteostasis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1449))

Abstract

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The ubiquitin–proteasome system is the major pathway for intracellular protein degradation in eukaryotic cells. Due to the large number of genes dedicated to the ubiquitin–proteasome system, mapping degradation pathways for short lived proteins is a daunting task, in particular in mammalian cells that are not genetically tractable as, for instance, a yeast model system. Here, we describe a method relying on high-throughput cellular imaging of cells transfected with a targeted siRNA library to screen for components involved in degradation of a protein of interest. This method is a rapid and cost-effective tool which is also highly applicable for other studies on gene function.

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References

  1. Finley D (2009) Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu Rev Biochem 78:477–513

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Reyes-Turcu FE, Ventii KH, Wilkinson KD (2009) Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Annu Rev Biochem 78:363–397

    Article  CAS  PubMed  Google Scholar 

  3. van Wijk SJ, Timmers HT (2010) The family of ubiquitin-conjugating enzymes (E2s): deciding between life and death of proteins. FASEB J 24:981–993

    Article  PubMed  Google Scholar 

  4. Deshaies RJ, Joazeiro CA (2009) RING domain E3 ubiquitin ligases. Annu Rev Biochem 78:399–434

    Article  CAS  PubMed  Google Scholar 

  5. Brooks CL, Gu W (2011) p53 regulation by ubiquitin. FEBS Lett 585:2803–2809

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Christianson JC, Olzmann JA, Shaler TA, Sowa ME, Bennett EJ, Richter CM, Tyler RE, Greenblatt EJ, Harper JW, Kopito RR (2012) Defining human ERAD networks through an integrative mapping strategy. Nat Cell Biol 14:93–105

    Article  CAS  Google Scholar 

  7. Poulsen EG, Steinhauer C, Lees M, Lauridsen AM, Ellgaard L, Hartmann-Petersen R (2012) HUWE1 and TRIP12 collaborate in degradation of ubiquitin-fusion proteins and misframed ubiquitin. PLoS One 7:e50548

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Pegoraro G, Voss TC, Martin SE, Tuzmen P, Guha R, Misteli T (2012) Identification of Mammalian protein quality control factors by high-throughput cellular imaging. PLoS One 7:e31684

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Barrangou R (2014) RNA events. Cas9 targeting and the CRISPR revolution. Science 344:707–708

    Article  CAS  PubMed  Google Scholar 

  10. Konig R, Chiang CY, Tu BP, Yan SF, DeJesus PD, Romero A, Bergauer T, Orth A, Krueger U, Zhou Y, Chanda SK (2007) A probability-based approach for the analysis of large-scale RNAi screens. Nat Methods 4:847–849

    Article  PubMed  Google Scholar 

  11. Zhang XD (2007) A pair of new statistical parameters for quality control in RNA interference high-throughput screening assays. Genomics 89:552–561

    Article  CAS  PubMed  Google Scholar 

  12. Lundholt BK, Scudder KM, Pagliaro L (2003) A simple technique for reducing edge effect in cell-based assays. J Biomol Screen 8:566–570

    Article  CAS  PubMed  Google Scholar 

  13. Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman O, Guertin DA, Chang JH, Lindquist RA, Moffat J, Golland P, Sabatini DM (2006) Cell Profiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol 7:R100

    Article  PubMed  PubMed Central  Google Scholar 

  14. Birmingham A, Selfors LM, Forster T, Wrobel D, Kennedy CJ, Shanks E, Santoyo-Lopez J, Dunican DJ, Long A, Kelleher D, Smith Q, Beijersbergen RL, Ghazal P, Shamu CE (2009) Statistical methods for analysis of high-throughput RNA interference screens. Nat Methods 6:569–575

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Zhang JH, Chung TD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73

    Article  PubMed  Google Scholar 

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Acknowledgmevnts

The authors apologize to those whose work was not cited due to space constraints. S.V.N., E.G.P., and R.H.P. are supported financially by grants from the Lundbeck Foundation and the Danish Council for Independent Research (Natural Sciences). The authors would like to acknowledge networking support by the Proteostasis COST Action (BM1307).

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    Authors and Affiliations

    1. Department of Biology, Section for Biomolecular Sciences, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen N, 2200, Denmark

      Esben G. Poulsen, Sofie V. Nielsen & Rasmus Hartmann-Petersen Ph.D.

    2. Biotech Research and Innovation Centre, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen N, 2200, Denmark

      Elin J. Pietras, Jens V. Johansen & Cornelia Steinhauer

    Authors
    1. Esben G. Poulsen
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    2. Sofie V. Nielsen
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    3. Elin J. Pietras
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    4. Jens V. Johansen
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    5. Cornelia Steinhauer
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    6. Rasmus Hartmann-Petersen Ph.D.
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    Corresponding author

    Correspondence to Rasmus Hartmann-Petersen Ph.D. .

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    Editors and Affiliations

    1. Computational and Experimental Biology Group Department of Health Promotion and Chronic Diseases, National Health Institute Dr. Ricardo Jorge, INSA, I.P., Lisboa, Portugal

      Rune Matthiesen

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    Poulsen, E.G., Nielsen, S.V., Pietras, E.J., Johansen, J.V., Steinhauer, C., Hartmann-Petersen, R. (2016). High-Throughput siRNA Screening Applied to the Ubiquitin–Proteasome System. In: Matthiesen, R. (eds) Proteostasis. Methods in Molecular Biology, vol 1449. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3756-1_28

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    • DOI: https://doi.org/10.1007/978-1-4939-3756-1_28

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    • Publisher Name: Humana Press, New York, NY

    • Print ISBN: 978-1-4939-3754-7

    • Online ISBN: 978-1-4939-3756-1

    • eBook Packages: Springer Protocols

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