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Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis

Nature Protocols volume 9, pages 263–293 (2014)Cite this article

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An Erratum to this article was published on 25 February 2016

This article has been updated

Abstract

The identification of sites where RNA-binding proteins (RNABPs) interact with target RNAs opens the door to understanding the vast complexity of RNA regulation. UV cross-linking and immunoprecipitation (CLIP) is a transformative technology in which RNAs purified from in vivo cross-linked RNA-protein complexes are sequenced to reveal footprints of RNABP:RNA contacts. CLIP combined with high-throughput sequencing (HITS-CLIP) is a generalizable strategy to produce transcriptome-wide maps of RNA binding with higher accuracy and resolution than standard RNA immunoprecipitation (RIP) profiling or purely computational approaches. The application of CLIP to Argonaute proteins has expanded the utility of this approach to mapping binding sites for microRNAs and other small regulatory RNAs. Finally, recent advances in data analysis take advantage of cross-link–induced mutation sites (CIMS) to refine RNA-binding maps to single-nucleotide resolution. Once IP conditions are established, HITS-CLIP takes ∼8 d to prepare RNA for sequencing. Established pipelines for data analysis, including those for CIMS, take 3–4 d.

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Figure 1: Overview of the HITS-CLIP protocol.
Figure 2: Sample CLIP autoradiograms (Step 34).
Figure 3: Sample RT-PCR from Ago CLIP experiments (Step 72).
Figure 4: Proportion of unique CLIP tags among all reads that were unambiguously mapped to the reference genome with regard to the size of the matched region (Step 97).
Figure 5: The positional profiles of each type of mutation relative to the 5′ end of reads (Step 105).
Figure 6: Enrichment of miR-124 seed site matches in sequences (±10 nt) around robust CIMS (FDR ≤0.
Figure 7: An example of a CIMS that precisely maps the Ago-miR-124-mRNA ternary complex (Step 112).

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Change history

  • 24 February 2016

     In the version of this article initially published, units were incorrectly reported in Step 2 of the Procedure. They have been changed from 'mJ cm2' to 'mJ per cm2' in four instances. In addition, units that previously read '1 = 0.1 J per m2' have been changed to '1 = 1 J per m2'. These errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We are indebted to Z. Mourelatos for sharing 2A8 antibody. We thank S. Dewell, C. Zhao and the entire staff of the Rockefeller University Genomics Resource Center for their expertise and support. We are grateful to sources of support for M.J.M. (Jane Coffin Childs Fund for Medical Research), C.Z. (US National Institutes of Health (NIH) grant no. K99/R00GM95713 and Simons Foundation grant no. 297990) and R.B.D. (grant nos. NIH NS081706 and NIH NS34389, the Simons Foundation and the Howard Hughes Medical Institute).

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Author notes

  1. Chaolin Zhang

    Present address: Present address: Department of Biochemistry and Molecular Biophysics, Columbia Initiative in Systems Biology, Center for Motor Neuron Biology and Disease, Columbia University, New York, New York, USA.,

  2. Michael J Moore and Chaolin Zhang: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Molecular Neuro-Oncology, The Rockefeller University,, New York, New York, USA

    Michael J Moore, Chaolin Zhang, Emily Conn Gantman, Aldo Mele, Jennifer C Darnell & Robert B Darnell

  2. Howard Hughes Medical Institute, The Rockefeller University, New York, New York, USA.,

    Robert B Darnell

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Contributions

M.J.M. wrote the sections of the manuscript pertaining to the experimental protocol and produced the data in Figure 2. C.Z. developed the methods and wrote manuscript sections pertaining to the bioinformatic protocol, including the analyses in Figures 4,5,6,7. E.C.G. produced the data in Figure 3a. A.M. produced the data in Figure 3b. J.C.D. contributed to writing sections of the experimental protocol. R.B.D. contributed to writing all sections and directed the development of all experimental and bioinformatic methods described here.

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Correspondence to Robert B Darnell.

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Moore, M., Zhang, C., Gantman, E. et al. Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis. Nat Protoc 9, 263–293 (2014). https://doi.org/10.1038/nprot.2014.012

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