capCLIP: a new tool to probe protein synthesis in human cells through capture and identification of the eIF4E-mRNA interactome.

Abstract

Translation of eukaryotic mRNAs starts with binding of the m7G cap to the protein eIF4E followed by recruitment of other translation initiation factors. eIF4Es essential role in translation suggests the cellular eIF4E-mRNA interactome (or eIF4E cap-ome) may serve as a faithful proxy of cellular translational activity. Here we describe capCLIP, a novel method to systematically capture and quantify the eIF4E cap-ome. To validate capCLIP, we identified the cap-omes in human cells {+/-} the partial mTORC1 inhibitor rapamycin. As expected, TOP (terminal oligopyrimidine) mRNA representation is systematically reduced in rapamycin-treated cells. capCLIP tag data permits refinement of a 7-nucleotide TOP motif (5'-CUYUYYC-3'). We also apply capCLIP to probe the consequences of phosphorylation of eIF4E, whose function had remained unclear. eIF4E phosphorylation drives an overall reduction in eIF4E-mRNA association; strikingly, mRNAs most sensitive to phosphorylation possess short 5'-UTRs. capCLIP provides a sensitive and comprehensive measure of cellular translational activity. We foresee its application as a high-throughput way to assess translation in contexts not amenable to existing methodologies.