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Identifying Protein Interactomes of Target RNAs Using HyPR-MS

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Post-Transcriptional Gene Regulation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2404))

Abstract

RNA–protein interactions are integral to maintaining proper cellular function and homeostasis, and the disruption of key RNA–protein interactions is central to many disease states. HyPR-MS (hybridization purification of RNA–protein complexes followed by mass spectrometry) is a highly versatile and efficient technology which enables multiplexed discovery of specific RNA–protein interactomes. This chapter provides extensive guidance for successful application of HyPR-MS to the system and target RNA(s) of interest, as well as a detailed description of the fundamental HyPR-MS procedure, including: (1) experimental design of controls, capture oligonucleotides, and qPCR assays; (2) formaldehyde cross-linking of cell culture; (3) cell lysis and RNA solubilization; (4) isolation of target RNA(s); (5) RNA purification and RT-qPCR analysis; (6) protein preparation and mass spectrometric analysis; and (7) mass spectrometric data analysis.

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  • 02 February 2022

    A correction has been published.

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Acknowledgments

This work was supported by NIH-NCI grant R01CA193481. K.B.H. was supported in part by the National Human Genome Research Institute grant to the Genomic Science Training Program, 5T32HG002760. R.M.M. was supported in part by the NIH Chemistry-Biology Interface Training Grant, T32GM008505. The authors would like to thank members of the Smith lab for helpful discussions and guidance in the development of HyPR-MS. The figures in this chapter were created with BioRender.com.

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Authors and Affiliations

  1. Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA

    Katherine B. Henke, Rachel M. Miller, Rachel A. Knoener, Mark Scalf, Michele Spiniello & Lloyd M. Smith

  2. Immuno-Hematology and Transfusion Medicine, Cardarelli Hospital, Naples, Italy

    Michele Spiniello

Authors
  1. Katherine B. Henke
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  2. Rachel M. Miller
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  3. Rachel A. Knoener
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  4. Mark Scalf
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  5. Michele Spiniello
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  6. Lloyd M. Smith
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Corresponding author

Correspondence to Lloyd M. Smith .

Editor information

Editors and Affiliations

  1. Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy

    Erik Dassi

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Henke, K.B., Miller, R.M., Knoener, R.A., Scalf, M., Spiniello, M., Smith, L.M. (2022). Identifying Protein Interactomes of Target RNAs Using HyPR-MS. In: Dassi, E. (eds) Post-Transcriptional Gene Regulation. Methods in Molecular Biology, vol 2404. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1851-6_12

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  • DOI: https://doi.org/10.1007/978-1-0716-1851-6_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1850-9

  • Online ISBN: 978-1-0716-1851-6

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