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

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Item Type:	Article
Title:	In vivo and transcriptome-wide identification of RNA binding protein target sites
Creators Name:	Jungkamp, A.C., Stoeckius, M., Mecenas, D., Gruen, D., Mastrobuoni, G., Kempa, S. and Rajewsky, N.
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.
Keywords:	Binding Sites, Computational Biology, Cross-Linking Reagents, Helminth RNA, Immunoprecipitation, Messenger RNA, RNA-Binding Proteins, Transcriptome, Animals, Caenorhabditis elegans
Source:	Molecular Cell
ISSN:	1097-2765
Publisher:	Cell Press
Volume:	44
Number:	5
Page Range:	828-840
Date:	9 December 2011
Official Publication:	https://doi.org/10.1016/j.molcel.2011.11.009
PubMed:	View item in PubMed

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