Article | Published:

Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP)

Nature Methods volume 13, pages 508514 (2016) | Download Citation

Abstract

As RNA-binding proteins (RBPs) play essential roles in cellular physiology by interacting with target RNA molecules, binding site identification by UV crosslinking and immunoprecipitation (CLIP) of ribonucleoprotein complexes is critical to understanding RBP function. However, current CLIP protocols are technically demanding and yield low-complexity libraries with high experimental failure rates. We have developed an enhanced CLIP (eCLIP) protocol that decreases requisite amplification by 1,000-fold, decreasing discarded PCR duplicate reads by 60% while maintaining single-nucleotide binding resolution. By simplifying the generation of paired IgG and size-matched input controls, eCLIP improves specificity in the discovery of authentic binding sites. We generated 102 eCLIP experiments for 73 diverse RBPs in HepG2 and K562 cells (available at https://www.encodeproject.org), demonstrating that eCLIP enables large-scale and robust profiling, with amplification and sample requirements similar to those of ChIP-seq. eCLIP enables integrative analysis of diverse RBPs to reveal factor-specific profiles, common artifacts for CLIP and RNA-centric perspectives on RBP activity.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Gene Expression Omnibus

References

  1. 1.

    , & A census of human RNA-binding proteins. Nat. Rev. Genet. 15, 829–845 (2014).

  2. 2.

    , , & RNA-binding proteins in Mendelian disease. Trends. Genet. 29, 318–327 (2013).

  3. 3.

    , , & RNA-binding proteins in neurodegeneration: Seq and you shall receive. Trends Neurosci. 38, 226–236 (2015).

  4. 4.

    et al. CLIP identifies Nova-regulated RNA networks in the brain. Science 302, 1212–1215 (2003).

  5. 5.

    et al. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141, 129–141 (2010).

  6. 6.

    et al. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat. Struct. Mol. Biol. 17, 909–915 (2010).

  7. 7.

    et al. Simultaneous generation of many RNA-seq libraries in a single reaction. Nat. Methods 12, 323–325 (2015).

  8. 8.

    et al. iCLIP: protein-RNA interactions at nucleotide resolution. Methods 65, 274–287 (2014).

  9. 9.

    et al. An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells. Nat. Struct. Mol. Biol. 16, 130–137 (2009).

  10. 10.

    et al. FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. Cell 146, 247–261 (2011).

  11. 11.

    et al. Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges. Nat. Struct. Mol. Biol. 20, 1434–1442 (2013).

  12. 12.

    et al. HITS-CLIP and integrative modeling define the Rbfox splicing-regulatory network linked to brain development and autism. Cell Rep. (2014).

  13. 13.

    et al. A multiprotein occupancy map of the mRNP on the 3′ end of histone mRNAs. RNA 21, 1943–1965 (2015).

  14. 14.

    , , & BackCLIP: a tool to identify common background presence in PAR-CLIP datasets. Bioinformatics (2015).

  15. 15.

    & Advancing the functional utility of PAR-CLIP by quantifying background binding to mRNAs and lncRNAs. Genome Biol. 15, R2 (2014).

  16. 16.

    , , & Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. Proc. Natl. Acad. Sci. USA 97, 14085–14090 (2000).

  17. 17.

    et al. PeakSeq enables systematic scoring of ChIP-seq experiments relative to controls. Nat. Biotechnol. 27, 66–75 (2009).

  18. 18.

    , , & Measuring reproducibility of high-throughput experiments. Ann. Appl. Stat. 5, 1752–1779 (2011).

  19. 19.

    et al. Resources for the comprehensive discovery of functional RNA elements. Mol. Cell (2016).

  20. 20.

    et al. A bifunctional protein regulates mitochondrial protein synthesis. Nucleic Acids Res. 42, 5483–5494 (2014).

  21. 21.

    & Prp8 protein: at the heart of the spliceosome. RNA 11, 533–557 (2005).

  22. 22.

    , , & SPF30 is an essential human splicing factor required for assembly of the U4/U5/U6 tri-small nuclear ribonucleoprotein into the spliceosome. J. Biol. Chem. 276, 31142–31150 (2001).

  23. 23.

    , & The human mitochondrial transcriptome and the RNA-binding proteins that regulate its expression. Wiley Interdiscip. Rev. RNA. 3, 675–695 (2012).

  24. 24.

    & A day in the life of the spliceosome. Nat. Rev. Mol. Cell Biol. 15, 108–121 (2014).

  25. 25.

    et al. LARP7 is a stable component of the 7SK snRNP while P-TEFb, HEXIM1 and hnRNP A1 are reversibly associated. Nucleic Acids Res. 36, 2219–2229 (2008).

  26. 26.

    et al. The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3. Nature 521, 232–236 (2015).

  27. 27.

    et al. Systematic discovery of Xist RNA binding proteins. Cell 161, 404–416 (2015).

  28. 28.

    et al. The A-repeat links ASF/SF2-dependent Xist RNA processing with random choice during X inactivation. Nat. Struct. Mol. Biol. 17, 948–954 (2010).

  29. 29.

    et al. Regulation of MALAT1 expression by TDP43 controls the migration and invasion of non-small cell lung cancer cells in vitro. Biochem. Biophys. Res. Commun. 465, 293–298 (2015).

  30. 30.

    et al. The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation. Mol. Cell 39, 925–938 (2010).

Download references

Acknowledgements

The authors would like to thank members of the Yeo lab (particularly S. Aigner and S. Markmiller) as well as colleagues J. Van Nostrand, Y. Kobayashi, B.R. Graveley and C.B. Burge for critical reading of the manuscript, and M. Blanco with early method development. This work was supported by grants from the US National Institutes of Health (HG004659, U54HG007005 and NS075449 to G.W.Y.), and by the US National Institutes of Health Director's Early Independence Award (DP5OD012190) and funds from the California Institute of Technology to M.G. We would also like to thank Ionis Pharmaceuticals for sharing reagents. E.L.V.N. is a Merck Fellow of the Damon Runyon Cancer Research Foundation (DRG-2172-13). G.W.Y. is an Alfred P. Sloan Research Fellow. G.A.P. is supported by the National Science Foundation Graduate Research Fellowship.

Author information

Affiliations

  1. Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California, USA.

    • Eric L Van Nostrand
    • , Gabriel A Pratt
    • , Chelsea Gelboin-Burkhart
    • , Mark Y Fang
    • , Balaji Sundararaman
    • , Steven M Blue
    • , Thai B Nguyen
    • , Keri Elkins
    • , Rebecca Stanton
    •  & Gene W Yeo
  2. Stem Cell Program, University of California at San Diego, La Jolla, California, USA.

    • Eric L Van Nostrand
    • , Gabriel A Pratt
    • , Chelsea Gelboin-Burkhart
    • , Mark Y Fang
    • , Balaji Sundararaman
    • , Steven M Blue
    • , Thai B Nguyen
    • , Keri Elkins
    • , Rebecca Stanton
    •  & Gene W Yeo
  3. Institute for Genomic Medicine, University of California at San Diego, La Jolla, California, USA.

    • Eric L Van Nostrand
    • , Gabriel A Pratt
    • , Chelsea Gelboin-Burkhart
    • , Mark Y Fang
    • , Balaji Sundararaman
    • , Steven M Blue
    • , Thai B Nguyen
    • , Keri Elkins
    • , Rebecca Stanton
    •  & Gene W Yeo
  4. Bioinformatics and Systems Biology Graduate Program, University of California at San Diego, La Jolla, California, USA.

    • Gabriel A Pratt
    •  & Gene W Yeo
  5. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA.

    • Alexander A Shishkin
    • , Christine Surka
    •  & Mitchell Guttman
  6. Ionis Pharmaceuticals, Carlsbad, California, USA.

    • Frank Rigo
  7. Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

    • Gene W Yeo
  8. Molecular Engineering Laboratory, A*STAR, Singapore.

    • Gene W Yeo

Authors

  1. Search for Eric L Van Nostrand in:

  2. Search for Gabriel A Pratt in:

  3. Search for Alexander A Shishkin in:

  4. Search for Chelsea Gelboin-Burkhart in:

  5. Search for Mark Y Fang in:

  6. Search for Balaji Sundararaman in:

  7. Search for Steven M Blue in:

  8. Search for Thai B Nguyen in:

  9. Search for Christine Surka in:

  10. Search for Keri Elkins in:

  11. Search for Rebecca Stanton in:

  12. Search for Frank Rigo in:

  13. Search for Mitchell Guttman in:

  14. Search for Gene W Yeo in:

Contributions

E.L.V.N., A.A.S., M.G., and G.W.Y. conceived the study. E.L.V.N., A.A.S., and C.S. developed the eCLIP methodology. E.L.V.N., C.G.-B., and S.M.B. performed 293T eCLIP and RBFOX2 knockdown experiments. F.R. provided antisense oligonucleotides (ASOs) and M.Y.F. performed ASO experiments. C.G.-B., B.S., S.M.B., T.B.N., K.E., and R.S. performed K562 and HepG2 eCLIP experiments. E.L.V.N. and G.A.P. performed computational analyses. E.L.V.N. and G.W.Y. wrote the manuscript.

Competing interests

F.R. is a paid employee of Ionis Pharmaceuticals.

Corresponding author

Correspondence to Gene W Yeo.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15, Supplementary Table 3, and Supplementary Protocol 1 and 2

Excel files

  1. 1.

    Supplementary Table 1

    Public CLIP dataset listing and associated read mapping values.

  2. 2.

    Supplementary Table 2

    eCLIP experiments deposited at the ENCODE Data Coordination Center, and associated read mapping values.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nmeth.3810

Further reading