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Mapping in vivo protein-RNA interactions at single-nucleotide resolution from HITS-CLIP data

Nature Biotechnology volume 29, pages 607–614 (2011)Cite this article

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Abstract

Mammalian RNA complexity is regulated through interactions of RNA-binding proteins (RBPs) with their target transcripts. High-throughput sequencing together with UV-crosslinking and immunoprecipitation (HITS-CLIP) is able to globally map RBP-binding footprint regions at a resolution of ∼30-60 nucleotides. Here we describe a systematic way to analyze HITS-CLIP data to identify exact crosslink sites, and thereby determine protein-RNA interactions at single-nucleotide resolution. We found that reverse transcriptase used in CLIP frequently skips the crosslinked amino-acid-RNA adduct, resulting in a nucleotide deletion. Genome-wide analysis of these crosslinking-induced mutation sites (CIMS) in HITS-CLIP data for Nova and Argonaute (Ago) proteins in mouse brain tissue revealed deletions in ∼8–20% of mRNA tags, which mapped to Nova and Ago binding sites on mRNA or miRNA. CIMS analysis provides a general and more precise means of mapping protein-RNA interactions than currently available methods and insight into the biochemical properties of such interactions in living tissues.

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Figure 1: Overview of CIMS analysis.
Figure 2: Crosslinking induces deletions, but not substitutions, that precisely map Nova-mRNA interactions.
Figure 3: Frequency of CIMS in CLIP tag clusters and association of CIMS with Nova-regulated alternative exons.
Figure 4: CIMS analysis refines the Nova binding motif.
Figure 5: Crosslinking induces deletions, but not substitutions, that precisely map Ago-mRNA and Ago-miRNA interaction sites.

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Acknowledgements

We thank J. Ule and all Darnell laboratory members for helpful discussion, and M.A. Frias, J. Luna, C.B. Marney and Y. Yuan for critical reading of the manuscript. This work was supported by grants from the National Institutes of Health (NS34389 to R.B.D. and K99GM95713 to C.Z.), and the Rockefeller University Hospital CTSA (UL1 RR024143). R.B.D. is a Howard Hughes Medical Institute Investigator.

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Authors and Affiliations

  1. Laboratory of Molecular Neuro-Oncology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York, USA

    Chaolin Zhang & Robert B Darnell

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  1. Chaolin Zhang
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Contributions

C.Z. and R.B.D. conceived and designed the study; C.Z. performed the research; C.Z. and R.B.D. analyzed the data and wrote the paper.

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Correspondence to Chaolin Zhang or Robert B Darnell.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1 and 5 and Supplementary Figures 1–6 (PDF 543 kb)

Supplementary Table 2

List of Nova CIMS (FDR ≤ 0.001) (XLS 2925 kb)

Supplementary Table 3

Summary of CIMS in Nova target cassette exons (XLS 72 kb)

Supplementary Table 4

List of Ago mRNA CIMS (FDR ≤ 0.001) (XLS 155 kb)

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Zhang, C., Darnell, R. Mapping in vivo protein-RNA interactions at single-nucleotide resolution from HITS-CLIP data. Nat Biotechnol 29, 607–614 (2011). https://doi.org/10.1038/nbt.1873

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