In vivo and treanscritopme-wide identification of RNA-binding protein target 
sites

Jungkamp, Anna-Carina; Stoeckius, Marlon; Mecenas, Desirea; Grün, Dominic; Mastrobuoni, Guido; Kempa, Stefan; Rajewsky, Nikolaus

doi:org/10.1016/j.molcel.2011.11.009

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In vivo and treanscritopme-wide identification of RNA-binding protein target sites

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Grün, Dominic
Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

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Jungkamp, A.-C., Stoeckius, M., Mecenas, D., Grün, D., Mastrobuoni, G., Kempa, S., et al. (2011). In vivo and treanscritopme-wide identification of RNA-binding protein target sites. Molecular Cell, 44, 828-840. doi:org/10.1016/j.molcel.2011.11.009.

Cite as: https://hdl.handle.net/21.11116/0000-0004-F955-4

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.