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RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome

Ian M Silverman123, Fan Li124, Anissa Alexander123, Loyal Goff56, Cole Trapnell56, John L Rinn567 and Brian D Gregory1234*

Author Affiliations

1 Department of Biology, University of Pennsylvania, 433 S. University Ave, Philadelphia, PA 19104, USA

2 PENN Genome Frontiers Institute, University of Pennsylvania, Philadelphia, PA 19104, USA

3 Cell and Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, PA 19104, USA

4 Genomics and Computational Biology Graduate Group, University of Pennsylvania, Philadelphia, PA 19104, USA

5 Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA

6 Broad Institute, Cambridge, MA 02142, USA

7 Beth Israel Deaconess Medical Center, Boston, MA 02215, USA

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Genome Biology 2014, 15:R3  doi:10.1186/gb-2014-15-1-r3

Published: 7 January 2014


Although numerous approaches have been developed to map RNA-binding sites of individual RNA-binding proteins (RBPs), few methods exist that allow assessment of global RBP–RNA interactions. Here, we describe PIP-seq, a universal, high-throughput, ribonuclease-mediated protein footprint sequencing approach that reveals RNA-protein interaction sites throughout a transcriptome of interest. We apply PIP-seq to the HeLa transcriptome and compare binding sites found using different cross-linkers and ribonucleases. From this analysis, we identify numerous putative RBP-binding motifs, reveal novel insights into co-binding by RBPs, and uncover a significant enrichment for disease-associated polymorphisms within RBP interaction sites.