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RNA. 2015 Jan;21(1):135-43. doi: 10.1261/rna.047803.114. Epub 2014 Nov 19.

Dissecting noncoding and pathogen RNA-protein interactomes.

Author information

1
Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California 94305, USA.
2
Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California 94305, USA Department of Bioengineering, Stanford University, Stanford, California 94305, USA.
3
Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, California 92697, USA.
4
Department of Microbiology and Immunology, McGill University, Montréal, QC H3A 2B4, Canada.
5
Department of Bioengineering, Stanford University, Stanford, California 94305, USA Department of Applied Physics, Stanford University, Stanford, California 94305, USA Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA.
6
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305, USA.
7
Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California 94305, USA Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA howchang@stanford.edu.

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.

KEYWORDS:

RNA–protein interactions; genomics; noncoding RNA; virology

PMID:
25411354
PMCID:
PMC4274633
DOI:
10.1261/rna.047803.114
[Indexed for MEDLINE]
Free PMC Article

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