title: Dissecting noncoding and pathogen RNA-protein interactomes.
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.
authors.affiliation: Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California 94305, USA.
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Flynn
authors.affiliation: Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California 94305, USA Department of Bioengineering, Stanford University, Stanford, California 94305, USA.
authors.forename: Lance
authors.initials: L
authors.lastname: Martin
authors.affiliation: Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, California 92697, USA.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Spitale
authors.affiliation: Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California 94305, USA.
authors.forename: Brian T
authors.initials: BT
authors.lastname: Do
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montréal, QC H3A 2B4, Canada.
authors.forename: Selena M
authors.initials: SM
authors.lastname: Sagan
authors.affiliation: Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California 94305, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Zarnegar
authors.affiliation: Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California 94305, USA.
authors.forename: Kun
authors.initials: K
authors.lastname: Qu
authors.affiliation: Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California 94305, USA.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Khavari
authors.affiliation: 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.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Quake
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Sarnow
authors.affiliation: 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.
authors.forename: Howard Y
authors.initials: HY
authors.lastname: Chang
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D016384:Consensus Sequence; D020869:Gene Expression Profiling; D016174:Hepacivirus / Q000502:physiology; D054884:Host-Pathogen Interactions; D006801:Humans; D047468:Immunoprecipitation; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D022661:RNA, Untranslated / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D059467:Transcriptome
chemical_list: D012333:RNA, Messenger; D022661:RNA, Untranslated; D012367:RNA, Viral; D016601:RNA-Binding Proteins
keywords: RNA–protein interactions; genomics; noncoding RNA; virology