Identifying DAP5 mRNA translation and direct-binding targets in human embryonic stem cells (hESCs) using Ribo-seq and CLIP-seq.

The eIF4G1 DAP5 homolog is involved in several non-canonical translation initiation mechanisms. We applied ribosome profiling and found that DAP5 translationally activated mRNAs are enriched with translated uORFs. We also performed a genome-wide UV-cross linking immunoprecipitation screen (CLIP-seq) for identifying mRNAs that directly interact with DAP5 in hESCs. We found 959 bound mRNAs common to both Abs (out of 1073 and 2152 enriched mRNAs identified by CS and MBL antibodies, respectively). Overall design: To asses DAP5 translation profile we performed ribosome profiling on DAP5-KD and NT control hESCs. The libraries were prepared from cells treated with the translation elongation inhibitor cycloheximide (CHX). In parallel, we used a tailored RNA-seq protocol to quantify RNA levels. For the CLIP-seq profiling H9 cells were UV cross-linked, lysed and treated with DNase. DAP5-RNA complexes were pulled down using 2 specific antibodies (Cell Signaling, 5169 or MBL, RN003P). The DAP5 bound mRNAs as well as the total RNA were used for libraries preparation.