Role of DDX3X in translation regulation in acute ER stress [mouse]

In eukaryotes, regulation of mRNA translation initiation greatly impacts gene expression, and is critical for cellular stress responses. DDX3X is a ubiquitous DEAD-box RNA helicase whose precise role in 5´ UTR scanning and start codon decoding in non-stressed and stressed cells is still elusive. Here we show that DDX3X engages with thousands of mRNAs as part of the 48S scanning complex, simultaneously acting to promote or suppress translation of select mRNAs in non-stressed conditions, and switches this regulation in opposite directions to establish a stress response translational program upon acute ER stress. We find distinct DDX3X binding patterns of differentially regulated mRNAs, which lead us to identify N4-acetylation of cytidines surrounding the start codon as a crucial feature for DDX3X-mediated selective regulation. Our findings highlight a novel co-dependence between an RNA helicase and a post-transcriptional modification in regulating mRNA translation Overall design: Mouse Embryonic Fibroblasts were grown to ~80% confluency and treated with control (DMSO) or Thapsigargin (400 nM) containing full medium for 1 h to induce acute ER stress. The cells where then collected by trypsinization and kept in ice-cold HBSS during UV crosslinking (254 nm, 3x400 mJ/cm2). To ensure that the procedure is not inducing stress in the control cells, we checked levels of eIF2a-P and ATF4 by western blot, and performed DDX3X immunofluorescence to observe possible induction of stress granules. For the IP step our own custom rabbit polyclonal DDX3X antibody was used. The rest of the CLIP-Seq procedure was performed as previously described (Vourekas and Mourelatos, 2014).