title: In vivo and transcriptome-wide identification of RNA binding protein target sites.
abstract: Animal mRNAs are regulated by hundreds of RNA binding proteins (RBPs). The identification of RBP targets is crucial for understanding their function. A recent method, PAR-CLIP, uses photoreactive nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. Here, we establish iPAR-CLIP (in vivo PAR-CLIP) to determine, at nucleotide resolution, transcriptome-wide binding sites of GLD-1, a conserved, germline-specific translational repressor in C. elegans. We identified 439 reproducible target mRNAs and demonstrate an excellent dynamic range of target detection by iPAR-CLIP. Upon GLD-1 knockdown, protein but not mRNA expression of the 439 targets was specifically upregulated, demonstrating functionality. Finally, we discovered strongly conserved GLD-1 binding sites near the start codon of target genes. These sites are functional in vitro and likely confer strong repression in vivo. We propose that GLD-1 interacts with the translation machinery near the start codon, a so-far-unknown mode of gene regulation in eukaryotes.
authors.affiliation: Systems Biology of Gene Regulatory Elements, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.
authors.forename: Anna-Carina
authors.initials: AC
authors.lastname: Jungkamp
authors.forename: Marlon
authors.initials: M
authors.lastname: Stoeckius
authors.forename: Desirea
authors.initials: D
authors.lastname: Mecenas
authors.forename: Dominic
authors.initials: D
authors.lastname: Grün
authors.forename: Guido
authors.initials: G
authors.lastname: Mastrobuoni
authors.forename: Stefan
authors.initials: S
authors.lastname: Kempa
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Rajewsky
mesh_terms: D000818:Animals; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000235:genetics*; D019295:Computational Biology; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D047468:Immunoprecipitation; D018510:RNA, Helminth / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D059467:Transcriptome / Q000235:genetics*
chemical_list: D003432:Cross-Linking Reagents; D018510:RNA, Helminth; D012333:RNA, Messenger; D016601:RNA-Binding Proteins