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Comprehensive analysis of RNA-protein interactions by high-throughput sequencing–RNA affinity profiling

Nature Methods volume 11, pages 683688 (2014) | Download Citation

Abstract

RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing–RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E–binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E–binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.

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Acknowledgements

We thank R. Friedman (Institut Pasteur) for providing scripts to extract intensity data, B. Mohanty (Medical University of South Carolina) for providing vectors containing the tus gene, K. Szeto and D. Shalloway for advice on data analysis, W. Zipfel and A. Singh for help in understanding the optics of the GAIIx, C.T. Waters and H. Kwak for bioinformatics advice, the Cornell sequencing core facility for help in learning the GAIIx, and H. Craighead (Cornell University) and the members of the Lis lab for helpful discussions on experimental design and the manuscript. This work was supported by US National Institutes of Health grants GM090320 and DA030329 to J.T.L.

Author information

Author notes

    • Jacob M Tome
    •  & Abdullah Ozer

    These authors contributed equally to this work.

Affiliations

  1. Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.

    • Jacob M Tome
    • , Abdullah Ozer
    • , John M Pagano
    •  & John T Lis
  2. Illumina, Inc., San Diego, California, USA.

    • Dan Gheba
    •  & Gary P Schroth

Authors

  1. Search for Jacob M Tome in:

  2. Search for Abdullah Ozer in:

  3. Search for John M Pagano in:

  4. Search for Dan Gheba in:

  5. Search for Gary P Schroth in:

  6. Search for John T Lis in:

Contributions

Initial idea: A.O. and J.T.L. Experimental design: J.M.T., A.O., D.G., G.P.S. and J.T.L. Experimental implementation: J.M.T., A.O. and J.M.P. HiTS-RAP experiments and bioinformatics analysis: J.M.T. Project coordination: J.T.L. Paper writing: J.M.T., A.O., J.M.P. and J.T.L.

Competing interests

D.G. and G.P.S. are employees of Illumina, Inc.

Corresponding author

Correspondence to John T Lis.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–9 and Supplementary Notes 1–4

Excel files

  1. 1.

    Supplementary Table 1

    All GFPapt mutants measured by HiTS-RAP

  2. 2.

    Supplementary Table 2

    HiTS-RAP measured GFPapt mutants verified by HiTS-RAP

  3. 3.

    Supplementary Table 3

    All GFPapt single base substitution mutants measured by HiTS-RAP

  4. 4.

    Supplementary Table 4

    All GFPapt double base substitution mutants measured by HiTS-RAP

  5. 5.

    Supplementary Table 5

    All NELFapt mutants measured by HiTS-RAP

  6. 6.

    Supplementary Table 6

    All NELFapt single base substitution mutants measured by HiTS-RAP

  7. 7.

    Supplementary Table 7

    All NELFapt double base substitution mutants measured by HiTS-RAP

  8. 8.

    Supplementary Table 8

    Primers used in this work

Zip files

  1. 1.

    Supplementary Software

    XML recipe used for the GFPapt run, and the Python program used for processing data, through fitting Kd values.

About this article

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DOI

https://doi.org/10.1038/nmeth.2970

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