In Vivo and Transcriptome-wide Identification of RNA Binding Protein Target Sites

SummaryAnimal 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.

► iPAR-CLIP identifies binding sites of RNA binding proteins (RBPs) in C. elegans
► iPAR-CLIP identified hundreds of direct targets of the germline-specific RBP GLD-1
► Effects of GLD-1 knockdown on protein expression of targets confirm functionality
► Conserved 5′ UTR sites near start codons are bound by GLD-1 and functional in vitro