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High-sensitivity in situ capture of endogenous RNA-protein interactions in fixed cells and primary tissues

Author

Listed:
  • Qishan Liang

    (University of California San Diego
    University of California San Diego)

  • Tao Yu

    (University of California San Diego
    University of California San Diego
    University of California San Diego
    University of California San Diego)

  • Eric Kofman

    (University of California San Diego
    University of California San Diego
    University of California San Diego
    University of California San Diego)

  • Pratibha Jagannatha

    (University of California San Diego
    University of California San Diego
    University of California San Diego
    University of California San Diego)

  • Kevin Rhine

    (University of California San Diego
    University of California San Diego
    University of California San Diego)

  • Brian A. Yee

    (University of California San Diego
    University of California San Diego
    University of California San Diego
    University of California San Diego)

  • Kevin D. Corbett

    (University of California San Diego
    University of California San Diego
    University of California San Diego)

  • Gene W. Yeo

    (University of California San Diego
    University of California San Diego
    University of California San Diego
    University of California San Diego)

Abstract

RNA-binding proteins (RBPs) have pivotal functions in RNA metabolism, but current methods are limited in retrieving RBP-RNA interactions within endogenous biological contexts. Here, we develop INSCRIBE (IN situ Sensitive Capture of RNA-protein Interactions in Biological Environments), circumventing the challenges through in situ RNA labeling by precisely directing a purified APOBEC1-nanobody fusion to the RBP of interest. This method enables highly specific RNA-binding site identification across a diverse range of fixed biological samples such as HEK293T cells and mouse brain tissue and accurately identifies the canonical binding motifs of RBFOX2 (UGCAUG) and TDP-43 (UGUGUG) in native cellular environments. Applicable to any RBP with available primary antibodies, INSCRIBE enables sensitive capture of RBP-RNA interactions from ultra-low input equivalent to ~5 cells. The robust, versatile, and sensitive INSCRIBE workflow is particularly beneficial for precious tissues such as clinical samples, empowering the exploration of genuine RBP-RNA interactions in RNA-related disease contexts.

Suggested Citation

  • Qishan Liang & Tao Yu & Eric Kofman & Pratibha Jagannatha & Kevin Rhine & Brian A. Yee & Kevin D. Corbett & Gene W. Yeo, 2024. "High-sensitivity in situ capture of endogenous RNA-protein interactions in fixed cells and primary tissues," Nature Communications, Nature, vol. 15(1), pages 1-16, December.
  • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-50363-4
    DOI: 10.1038/s41467-024-50363-4
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    References listed on IDEAS

    as
    1. Longxing Cao & Brian Coventry & Inna Goreshnik & Buwei Huang & William Sheffler & Joon Sung Park & Kevin M. Jude & Iva Marković & Rameshwar U. Kadam & Koen H. G. Verschueren & Kenneth Verstraete & Sco, 2022. "Design of protein-binding proteins from the target structure alone," Nature, Nature, vol. 605(7910), pages 551-560, May.
    2. Meeli Mullari & Nicolas Fossat & Niels H. Skotte & Andrea Asenjo-Martinez & David T. Humphreys & Jens Bukh & Agnete Kirkeby & Troels K. H. Scheel & Michael L. Nielsen, 2023. "Characterising the RNA-binding protein atlas of the mammalian brain uncovers RBM5 misregulation in mouse models of Huntington’s disease," Nature Communications, Nature, vol. 14(1), pages 1-20, December.
    3. Hugo C. Medina-Munoz & Eric Kofman & Pratibha Jagannatha & Evan A. Boyle & Tao Yu & Krysten L. Jones & Jasmine R. Mueller & Grace D. Lykins & Andrew T. Doudna & Samuel S. Park & Steven M. Blue & Brodi, 2024. "Expanded palette of RNA base editors for comprehensive RBP-RNA interactome studies," Nature Communications, Nature, vol. 15(1), pages 1-17, December.
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