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PMID:22152485

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Citation

Jungkamp, AC, Stoeckius, M, Mecenas, D, Grün, D, Mastrobuoni, G, Kempa, S and Rajewsky, N (2011) In vivo and transcriptome-wide identification of RNA binding protein target sites. Mol. Cell 44:828-40

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.

Links

PubMed PMC3253457 Online version:10.1016/j.molcel.2011.11.009

Keywords

Animals; Binding Sites; Caenorhabditis elegans/genetics; Computational Biology; Cross-Linking Reagents/chemistry; Cross-Linking Reagents/metabolism; Immunoprecipitation; RNA, Helminth/genetics; RNA, Helminth/metabolism; RNA, Messenger/genetics; RNA, Messenger/metabolism; RNA-Binding Proteins/metabolism; Transcriptome/genetics

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

CAEEL:GLD1

involved_in

GO:0017148: negative regulation of translation

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

CAEEL:GLD1

enables

GO:0003730: mRNA 3'-UTR binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

CAEEL:GLD1

enables

GO:0048027: mRNA 5'-UTR binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete


See also

References

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