folder

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

Authors

  • A.C. Jungkamp
  • M. Stoeckius
  • D. Mecenas
  • D. Gruen
  • G. Mastrobuoni
  • S. Kempa
  • N. Rajewsky

Journal

  • Molecular Cell

Citation

  • Mol Cell 44 (5): 828-840

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.


DOI

doi:10.1016/j.molcel.2011.11.009