Dissecting noncoding and pathogen RNA–protein interactomes
- Ryan A. Flynn1,8,
- Lance Martin1,2,8,
- Robert C. Spitale3,
- Brian T. Do1,
- Selena M. Sagan4,
- Brian Zarnegar1,
- Kun Qu1,
- Paul A. Khavari1,
- Stephen R. Quake2,5,6,
- Peter Sarnow7 and
- Howard Y. Chang1,6
- 1Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California 94305, USA
- 2Department of Bioengineering, Stanford University, Stanford, California 94305, USA
- 3Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, California 92697, USA
- 4Department of Microbiology and Immunology, McGill University, Montréal, QC H3A 2B4, Canada
- 5Department of Applied Physics, Stanford University, Stanford, California 94305, USA
- 6Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA
- 7Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305, USA
- Corresponding author: howchang{at}stanford.edu
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↵8 These authors contributed equally and share first authorship.
Abstract
RNA–protein interactions are central to biological regulation. Cross-linking immunoprecipitation (CLIP)-seq is a powerful tool for genome-wide interrogation of RNA–protein interactomes, but current CLIP methods are limited by challenging biochemical steps and fail to detect many classes of noncoding and nonhuman RNAs. Here we present FAST-iCLIP, an integrated pipeline with improved CLIP biochemistry and an automated informatic pipeline for comprehensive analysis across protein coding, noncoding, repetitive, retroviral, and nonhuman transcriptomes. FAST-iCLIP of Poly-C binding protein 2 (PCBP2) showed that PCBP2-bound CU-rich motifs in different topologies to recognize mRNAs and noncoding RNAs with distinct biological functions. FAST-iCLIP of PCBP2 in hepatitis C virus-infected cells enabled a joint analysis of the PCBP2 interactome with host and viral RNAs and their interplay. These results show that FAST-iCLIP can be used to rapidly discover and decipher mechanisms of RNA–protein recognition across the diversity of human and pathogen RNAs.
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Footnotes
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Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.047803.114.
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Freely available online through the RNA Open Access option.
- Received August 21, 2014.
- Accepted October 27, 2014.
This article, published in RNA, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
