alexa In vivo and transcriptome-wide identification of RNA binding protein target sites.
Chemical Engineering

Chemical Engineering

Journal of Chromatography & Separation Techniques

Author(s): Jungkamp AC, Stoeckius M, Mecenas D, Grn D, Mastrobuoni G,

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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. Copyright © 2011 Elsevier Inc. All rights reserved.
This article was published in Mol Cell and referenced in Journal of Chromatography & Separation Techniques

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