• A Corrigendum to this article was published on 27 April 2018

This article has been updated

Abstract

RNA–protein interactions play numerous roles in cellular function and disease. Here we describe RNA–protein interaction detection (RaPID), which uses proximity-dependent protein labeling, based on the BirA* biotin ligase, to rapidly identify the proteins that bind RNA sequences of interest in living cells. RaPID displays utility in multiple applications, including in evaluating protein binding to mutant RNA motifs in human genetic disorders, in uncovering potential post-transcriptional networks in breast cancer, and in discovering essential host proteins that interact with Zika virus RNA. To improve the BirA*-labeling component of RaPID, moreover, a new mutant BirA* was engineered from Bacillus subtilis, termed BASU, that enables >1,000-fold faster kinetics and >30-fold increased signal-to-noise ratio over the prior standard Escherichia coli BirA*, thereby enabling direct study of RNA–protein interactions in living cells on a timescale as short as 1 min.

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Change history

  • 20 April 2018

    In the version of this Article originally published, an amino acid substitution introduced into the B. subtilis biotin ligase sequence was incorrectly written as R142G. The correct mutation is R124G. This error has been corrected in the HTML and PDF versions of the paper.

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Acknowledgements

We thank C. Adams, R. Leib and Vincent Coates Foundation Mass Spectrometry Laboratory, Stanford University Mass Spectrometry for help with mass spectrometry. We also thank P. Oikonomou and S. Tavazoie for providing position weight matrixes. We thank A. Fire, H. Chang, J. Elias, P. Sarnow, R. Flynn, J. Quinn, A. Bhaduri, A. Rubin and M. Kay for presubmission review. We thank L. Morcom and P. Bernstein for expert administrative assistance and members of the Khavari lab for helpful discussions. This work was supported by the US VA Office of Research and Development, by NIH AR49737, and NIH AR43799 (P.A.K.). The project described was supported by Award Number S10RR027425 from the National Center For Research Resources. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health.

Author information

Affiliations

  1. Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA.

    • Muthukumar Ramanathan
    • , Deepti S Rao
    • , Poornima H Neela
    • , Brian J Zarnegar
    • , Smarajit Mondal
    • , Joanna R Kovalski
    • , Zurab Siprashvili
    •  & Paul A Khavari
  2. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA.

    • Karim Majzoub
    •  & Jan E Carette
  3. Department of Neurosurgery, Stanford University School of Medicine, Stanford, California, USA.

    • Julien G Roth
    • , Hui Gai
    •  & Theo D Palmer
  4. Veterans Affairs Palo Alto Healthcare System, Palo Alto, California, USA.

    • Paul A Khavari

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Contributions

M.R. designed and executed experiments, analyzed the data and wrote the manuscript. K.M. designed and performed viral replication assays. J.G.R., H.G., D.S.R., J.R.K., P.H.N., B.J.Z., Z.S. and S.M. performed experiments. J.E.C. and T.D.P. supervised work. P.A.K. designed experiments, analyzed data, and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Paul A Khavari.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–12 and Supplementary Notes 1–2

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Protocol

    RaPID-Western

Excel files

  1. 1.

    Supplementary Table 2

    EDEN15 RaPID-MS in HEK293T cells.

  2. 2.

    Supplementary Table 3

    EDEN15 RaPID-MS in Huh7 cells.

  3. 3.

    Supplementary Table 4

    FTL IRE RaPID-MS in HEK293T cells.

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    Supplementary Table 5

    ZIKV 5′UTR RaPID-MS in HEK293T cells.

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    Supplementary Table 6

    ZIKV 3′UTR RaPID-MS in HEK293T cells.

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    Supplementary Table 7

    ZIKV UTR host proteins DAVID GO Term Analysis.

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    Supplementary Table 8

    Tissue Protein Level data for ZIKV UTR interacting proteins.

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    Supplementary Table 9

    Histone Stem Loop (UTRP11) RaPID-MS in HEK293T cells.

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    Supplementary Table 10

    p38_NRE RaPID-MS in HEK293T cells.

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    Supplementary Table 11

    PPP1R3C (UTRP30) RaPID-MS in HEK293T cells.

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    Supplementary Table 12

    Synthetic-EIR concatamer RaPID-MS in HEK293T cells.

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    Supplementary Table 13

    SM1v1 RaPID-MS in HEK293T cells.

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    Supplementary Table 14

    BASU EDEN15 RaPID-MS in HEK293T cells at 30 minutes.

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    Supplementary Table 15

    FTL IRE RaPID-MS in HEK293T cells at 30 minutes.

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    Supplementary Table 16

    List of Background proteins in HEK293T cells

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    Supplementary Table 17

    RNA motif sequences utilized in RaPID.

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    Supplementary Table 18

    Composition of buffers used in RaPID.

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DOI

https://doi.org/10.1038/nmeth.4601

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